BIOL 121 Notes on Powerpoint Slides
BIOL 121 Notes on Powerpoint Slides BIOL 121
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Date Created: 08/21/15
Reminder Discussion session this Monday at 9 am in Leidy 10 Weekend questions will be posted You can also nd more practice questions in the old exams posted on Canvas Enzymes are proteins that function as catalysts They are involved in virtually all cellular processes and affect many types 0f chemical reactions processes not caused by enzymes generally automatically happen due to the chemistry Reaction rate can Increase by as much as 1017fold eg Orotidine 5 phosphate decarboxylase reduces a 78 million year reaction time to 18 milliseconds Class Reaction Type Class Reaction Type 1 xidoreductases xidationreduction 4 I Yases Rcmm al 0 group reactions lrom or addition ofa group to a molecule tranSfer 0f 939 Often With H with rearrangement of electrons forms double bonds 2 Transferases Transfer of functional groups from one molecule to another 5 Isomerases Movement of a functional grotip within a molecule 3 l lydrolases Hydrolytic cleavage of one molecule into 6 Ligases Joining of two two molecules molecules to form a single molecule ase39 Table 61 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Examples of enzymecatalyzed reactions c Sucrase 39 CHZOH hydrolysis ll Sucrose Glucose C12H22011 C6H1206 common sugar breaks down sugar in milk CH20H CH30H CHon 0 O 0 H0 H H Lactase HO OH 0 H OH H H OH H 0H OH H H H20 H H OH H OH H OH Lactose DGalactose Fructose C6H1206 u honey CH20H o H H HO OH H OH H OH DGlucose lactose intolerant insufficient amounts of lactase gt water pulled into intestines and bacteria eat it aka e ooli gastrointestinal stress An enzyme catalytic cycle the basic principles 0 Substrates enter active site enzyme changes shape so its active site maximizes interaction with the substrates induced t d f SUBSTRATES S ENZYMESUBSTRATE COMPLEX ES 6 Active site is available for two new substrate molecules ENZYME E cquot Productsreleased 39 PRODUCTS P eSubstrates held in active site by weak interactions such as hydrogen bonds and ionic bonds Speci city for substrate 9 Active site and R groups of its amino acids can lower AG and speed up a reaction by acting as a template for substrate orientation stressing the substrates and stabilizing the transition state providing a favorable microenvironment participating directly in the catalytic reaction GSubstrates are converted into products The site on enzymes where substrates bind and chemistry occurs is the active site defense mechanism tears mucus gastrointestinal gt degrades cell walls of bacteria b Folded lysozyme a Unfolded lysozyme u The active site contains o o 8 3900 am 35 Binding pocket 0 00 O Asp52 o o 0 Catalytic Slte O 129 O O 1 0 0 C Active site H 1 Ala107110 3 O 100 40 Glu35 N 0 0 I39 1w by Q t e 0 039 70 o Ala107 3 s zzsquot 90 O O Q 50 two neg 39 80 g T 63 0 amino acids f quot Q r 4 A 0 0 p Asp52 quot May requnre something other 39000 than enzyme itself to work m contact pos charged Intermediate Cofactors Fig 62 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson o Coenzymes Prosthetic groups LOWER FREE ENERGY STATE Often vitamins Rate of reaction Enzymes like many proteins are sensitive to heat pH and other environmental conditions Optimal temperature for Optimal temperature for pH optimum a typical human enzyme a typical enzyme of for trypsin thermophilic E pH optimum heattolerant s for pepsin bacteria g 6 0 5 o O 20 40 60 80 1 00 0 1 2 3 4 5 Temperature 00 What actors determine this SBIISiIilliW Chemist Protein SII IIGIIII G Fig 64 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Enzyme kinetics describes the quantitative aspects of enzyme catalysis and reaction rates 39 HOW FAST a reaction occurs not if the reaction Will occur Typically talk about initial reaction rates VO at which product is being formed Enzymes only work if the reaction is already able to occur The if is determined by thermodynamics Reaction rates are in uenced by the environment and factors such as the concentrations of enzyme E substrates 8 products P and inhibitors X concentration of x The catalytic cycle Id if SUBSTRATES S ENZYMESUBSTRATE COMPLEX ES ENZYME E N PRODUCTS P The catalytic cycle can be modeled as a simple reaction SE ES PE Initial reaction rate Va is measured over a brief time at a particular E and initial 5 v0 is also called the initial velocity Note your text uses v instead of V0 for initial velocity For early times the concentration increases V0 IAPAt WWWJ approximately linearly nd atjnae V0 is determined from this short time interval leveling out equilibrium relative concentrations of p and 3 never reaches 0 V0 linear concentration Vo can neglect hte reverse rxn 0 time httpwww Wiley comcollegepratt 0471393878studentanimations enzymekinetics At early times S has not yet decreased enough during experiment to affect the rate of P formation P has not yet increased enough for the reverse reaction P E gt ES to be significant Determination of initial velocity Vo of enzymecatalyzed reaction as a function of substrate concentration 5 Equilibrium v s T 0 4 gto gt 395 3 396 a O 52 gt 3 m D E 51 Substrate concentration S Time gt de39ta 393 Can we understand the Initial rate V0 for each substrate concentration is determined from the slopes of the curves at early time points shape of this curve MichaelisMenten Kinetics SEEST PE Neglect reverse reaction and assume S is constant since measuring initial velocity At steady state ES is constant V S max quot0 Km S Maud Menten and Leonor Michaelis httpwwwjbcorgcontent27949coverexpansion Michaelis L and Menten ML 1913 Die kinetik der invertinwirkung Biochim 2 42 333369 Initial velocity v0 Michaelis Menten Kinetics MIA max 3 m x 3R gt Substrate concentration S KM Michaelis constant and Vm1X are constants that depend on the reaction rate constants which in turn depend on the speci c enzyme and environmental conditions Vm1X also depends on total enzyme concentration VIm1X is the maximal possible velocity for a given E MD Km is the value of S at which the rate is equal to 12 Vm1X Note Km has units of concentration 68 Becker World of 96 Cell 8277 ed Ham39m 62 61 2072 P65273072 Low substrate concentration If SltltKm then Km Sz1lt V Km 8 Km max 5 E S gt 5 E O F Vmax E s S E S E E E E g E E E Most of the enzymes are not bound to substrate l T Substrate concentration S Km 68 Bet 673 World of 96 Cell 8 ed Ham z39 62 52 2072 P6473071 At intermediate S each additional increase in S results in a smaller increase in V0 Initial velocity v0 MIA max 3 m x VmaXIS v 0 Km S s S S E s E E E E E S An increasing fraction of the enzyme is occupied with substrate ES Substrate concentration S I I I I I I I Km 68 Bet 673 World of 96 Cell 857 ed Ham z 62 52 2072 P6473071 When S Km VOIV Initial velocity v0 2 initial velocity is half maximal maX substrate This is the de nition of Km V Vmaxis VmaXKm Vmax 0 Km S 2Km 2 Vmax s S E s E S s E S E 1 S E S S E E Vmagtlt E S l E S S E E E E l Half of the enzymes are bound with l l Substrate concentration S Km 68 Bea 673 World of 96 Cell 8 ed Ham z39 62 52 2072 P6473072 When s1 gtgt Km high v0 v0 z Vm maximal rate under the given conditions Enzymes are saturated with substrate ES Vmaxis Vmaxisi V max Km S S V max 8 S S S E 2 S S E S 3 S B S E s E O E Vmax I S E s E S E S E E S s E c S E i s E s I S i Substrate concentration S Km 68 Bet 673 World of 96 Cell 8 ed Ham z 62 52 2072 P6473071 Ilow then can vnu increase quotmax Vmax IS proportlonal to the total enzyme concentratlon 11nreases Vim1X Increasing tota MichaelisMenten I I HBX total max LOW ELoml Same KM KM 51 Total enzyme concentration Etota 63 Beg 673 Wrd 0f 96 C6 8179 mi Hamil e 4 2072 1364730 7ij WWW wily 60777 coege pmz f 047 73938 7 8 yz ude f a imczz z39om M i fi Enzyme Inhibition Competitive Substrate inhibitor Substrate Noncompetitive Kinetics of competitive inhibition S E 4 ES gt E P l Competitive I Substrate inhibit0rv 10 No inhibitor 80 IOW 3 f E 60 I high A competitive inhibitor binds only to free g 40 I medium enzyme I competes with S The inhibitor 20 increases the apparent Km but does not change Vmax o 39 Substrate gt Biochemistry 7 ed Berg Tymoczko Stryer Note competitive inhibitors do not necessarily bind in the active site although they often do Kinetics of noncompetitive inhibition S EI gtEs gtEP Substrate 4 Noncompetitive s inhibitor 1 El 3 a ESI gt no 1 8 product 100 No inhibitor I low Relative rate I medium Substrate gt Biochemistry 7 ed Berg Tymoczko Stryer A noncompetitive inhibitor binds equally well to free enzyme and enzyme complexed with substrate I does not compete with 8 Consequently Vmax cannot be attained even at high substrate concentrations decreases the apparent Vmax but does not change Km Methotrexate an anticancer drug that is a competitive inhibitor of dihydrofolate reductase 0 OH o v OH DAN 3quot u o NH2 N N O OH Fi 39 39 W a H2N N N O l I 1 i O N 39 39 m axoncw 7 H E123 ow N N N OH 39mr39 III A I H DHF O M H2N N N H H Methotrexate MTX is a structural analog of dihydrofolate DHF a substrate for the enzyme dihydrofolate reductase that plays a central role in the bios nthesis y of purines and pyrimidines MTX binds DHFR 100fold more tightly than DHF MTX is used to treat some cancers and other conditions associated with DNA replication Competitive and noncompetitive inhibitors are examples of reversible inhibition Some inhibitors are irreversible eg by covalently modifying an enzyme Irreversible enzyme inhibition by the antibiotic penicillin Inhibition of growth of R Staphylococcus aureus by Penicilium mold OC H H NH CH3 R N 5 CH3 S ifelf Noaigma fCHs PenicillinCOOH x H 39 OH Ser Penicilloylenzyme complex enzymatically inactive Glycopeptide transpeptidase Bacterial cell walls contain Damino acids that form crosslinks through the action of a glycopeptide trans eptidase Penicillin binds this enzyme and covalently modi ies it to abolish catalytic activity irreversibly Allosteric effectors bind at sites away from the active site and change enzyme activity Allosteric enzymes have two different conformations one that is high affinity and one that has low affinity for the substrate 39 Effectors bind to an allosteric site or regulatory site 39 Effectors regulate by binding and stabilizing one conformation Active Allosteric Site VSubstrate Products site k 4 Highaffinity form of enzyme Allosteric 394 inhibitor Allosteric i Little or no product 1nh1b1tor formation Lowaffinity form of enzyme Active Allosteric site site Little or no product AHOStCI lC formation actlvator Lowaffinity form of enzyme 39 Allosteric O activator Substrate Pmducts s Highaffinity form of enzyme 676 Becker World of 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Note allosteric inhibitors can be either competitive or non competitive Membrane uidity is in uenced by sterols such as cholesterol that act as a uidity buffer hydroxl group polar aspect cholesterol sterol Gel phase solid phase gt cells die Regulation of fluidity Rigidity of cholesterol decreases uidity abOV the mdtl g t mp mtur disrupts the packing melt more CHz CH CHZ ke Changmg the VISCOS39ty easily gt like adding salt to the I I Bulklness of cholesterol prevents road Hydrogen bond oc39 oH phospholipids tails from packing together lowering the melting temperature Phospholipid lt Phospholipid Cholesterol w l O gt i gt i gt i K 7 75 Becker lVord 0f 2796 Cell 8279 ed H4707 62 4 2072 P661730 i i Cell membranes need to be 39es e39 39 in the fluid state to function 7 75 Beckerfr World of 96 Cell 8277 ed Ham z39 62 61 2072 PedVJO Plants make many sterols subject to large temp fluctuations gt sterols in bilayers regulate fluidity Membranes contain integral peripheral and lipidanchored proteins SURFACE p p I 9 w p 4 o o o Q o 9 9 c v q 1 o ov vv39r otn t r 39 51 6 Q t o v t o 9 v t Q v 1 h9Qloc f CQQQQQquot39JV OC10 39 yvooqov y v o o q o v 91 07vf39 g c v 9 v e v I o 9 v Q VVquot QQv VOP b v V o v w o v w v0 i v o v o v C Q i us Q v o s V 39 1 w V s vvquot ioqvooltw Ov vaiQQtQVQQOV DV uvqooo39o o o o v o o I v o o a Q o o o o o I u I o v oloooonf quot IOOOOII39PR III39IOOII IOO IOI39OOJIIOIOOI I r 39 I i I I F i 39 g I ooooooooo I o o o o o o o o o o o o o o o o o oar 4quot o o o o a o o o o o u c o o o v o o o o o o o a o o o Li INNER g GPI SURFACE 5 Iquot KOJ anchor a Integral b Singlepass c Multipass d Multi e Peripheral f Fatty acid or monotopic protein protein subunit membrane isoprenyl protein protein protein anchor Y J Y j Integral membrane proteins aSSOC with pid Lipidanchored two distinct head membrane proteins 779 Becker War510 96 Cell 8277 ed Hardin 62 61 2072 P66175072 p0ypeptides groups hydrophobic appendage Parts of membrane IOOPS around arid wants to hide embedded in 1 comes baCk leaflet Gram negative bacteria have 2 membranes plasma membrane and outer membranepermeable extra layer of protection gt antibiotic resistance beta barrels gt only in outer layer or when pathogens insert in plasma memb to kill cell Transmembrane domains beta sheet wrapped into a cylinder hole running down the middle leaking lipid b ayer 5231 Beta barrel Single Multiple alpha alpha helix helices Fig 1017 Molecular Biology of the Cell 4th edition Protein segments spanning the bilayer generally have non polar residues in contact with the hydrophobic chains of the lipids An example of an alpha helical transmembrane domain lt hydrophillic region EXTRACELLULAR 200 SPACE 51quot g polar amino acids are just there or making m 5 contact with polar amino acids of other ij 9 alpha helices The hydrophoblc core of my m3 3 395 blologlcal membranes are m a generally 3 4 mm tthk Q 8 0 ma 113E 39 0 rim i 0 3m CYTOSOL 2 mt O nonpolar A The uid mosaic model of cell membranes a phospholipid bilayer in the uid phase with proteins dispersed throughout 39 Lipids in each monolayer are in constant motion 39 Proteins are also able to move laterally within the membrane though some are anchored to internal structural elements Lipid rafts are membrane microdomains enriched in cholesterol sphingolipids and glycospingolipids more ordered than the surrounding 7 3 Biology 7277 eel Campbell and Reece 2005 Pearson Often CO tal l g many Slg ah g proteins Diffusion and Membrane Transport across the membrane Membranes provide a boundary between the interior and exterior of the cell and of compartments organelles within the cell In general concentrations of chemical species differ on the two sides of a membrane The difference in concentration across the membrane is called a concentration gradient or chemical gradient Many membranes also have an electrical potential difference across the two sides called a potential gradient The concentration gradient and the electrical potential gradient are together called the electrochemical gradient The electrical component will only affect charged solutes For charged molecules an electrical potential gradient is a driving force that moves positive ions from the high potential to the low potential side A concentration gradient exerts a similar effect on molecules charged or neutral When there is a concentration gradient of solute diffusion will give rise to a net movement from high to low concentrations Na gt huge energy barrier for sodium to cross theoretically very eventually it would reach equilibrium Membrane transport by simple diffusion the unassisted net movement of a solute across the membrane For uncharged solutes transport from higher to lower concentration the rate is proportional to the difference in concentration between the two sides of the membrane The membrane is selectively permeable Lipid bilayers have very low permeabilities to charged species In general with the exception of water membrane permeability increases with increasing solubility in nonpolar hydrocarbon like solvents hydrogen bond with itself parts the lipids 7 77 Biology 7277 ed Campbell and Reece 2005 P66175071 What if there is a concentration gradient of solute but the membrane is impermeable to the solute molecules In this case water will diffuse from the low to high concentration side This is called Osmosis Net diffusion of water across the membrane will continue until total solute concentrations are equal on the two sides or until a high pressure builds up Animals cells which lack a cell wall are particularly sensitive to changes in solute concentration outside the cell 8A7 Bea erfr LVord of 196 Cell 8277 ed H61m 13911 61 61 2072 P66173011 Osmotic pressure HYPERTONIC SOLUTION ISOTONIC SOLUTION HYPOTONIC SOLUTION Inside Outside of cell of cell ANIMAL CELL a Shriveled Normal Lysed 69 sze 70277 ed 5 61d6w61 61 61 2074 WH Free716111 Channel proteins and carrier proteins facilitate passive transport across membranes facilitated diffusion Channel proteins Carrier proteins Hydroph ic Channels 39 Carrier proteins bind solute molecules on one side of a through membrane membrane undergo a Route for solute conformation change and release the solute on the other side of the membrane 39 C2111 136 gated 39 Tend to have solute speci city catalyze process llow mlglll these nroteins he sneciiic Can have solute specificity Channel proteins transport solutes 1 faster than carrier proteins gt 39 1 7i Sol te Carner protem 1 A a Chann Solute 39 7 J5 Biology 7277 ed Campbell and Reeve 2005 Penman Carrier proteins can transport one or two different solutes OUTER Sb SURFACE S 3 Sb Sa tgg ia ltj 39 quot vi1tiiiif ZZ 0 INNER S SURFACE Sa Sb S b S Y J xa Y J Symport Antiport Y J Uniport Coupled transport Fzg 86 Beckeer LVord 0f 96 Cell 8277 ed Ham39m 62 52 2072 P65273072 Carrier proteins are analogous to enzymes No energy input is needed The carrier proteins lower the energy barrier for transport from one side of the membrane to the other Carriers transiently bind the carrier like enzymes binding a substrate leads to speci city for certain solutes gives rise to saturation at high solute concentration gt carrier protein at any instant is transporting a substrate competition inhibition by other solute species that are also substrates for the carrier Carriers can be modeled with Michaelis Menten kinetics Facilitated diffusion hyperbolic rate of diffusion Carrier protein 0 Q Q 7 J5 Biology 7 ed Campbell cmd Reece 2005 P65273072 V Simple diffusion linear A S solute concentration gradient A S outside i SidC Active transport is protein mediated movement against the electrochemical gradient Couples unfavorable transport Direct Indirect With ATP hydroly81s or some W W W H H other source of energy such as OUTSIDE H quot1H H OF CELL Hquot Hquot favorable transport of another o V o o o 7 o o o A 10 s O 390 v0 10 o o o 390 974 t 1 7 39 o o o v o 0 quot00000 39 n o v l I 39 a o o 1 gt i 39 l I i V V gt O OOOOOOCO OO OOOOIi O O I 39 O 3 molecule Important for many cellular y O O C O O O O O O O O O INSIDE OF CELL y l I I O O b O O O O f 3 l functions such as Uptake of essential nutrients Removal of wastes S 0 s S M i tC a CC Of O 673 sze 70277 ed Sadam 62 52 2074 WH Freeman equilibrium concentrations of Inir rnrrn mrrrn39rr certam Ions 6g high potassium d ect t a spo te 8 ca be sy po te s o a t1po te 8 inside and low sodium outside Active transporters are also called pumps Simple diffusion facilitated diffusion and active transport move molecules across membranes in different ways Table 8l Comparison of Simple Diffusion Facilitated Diffusion and Active Transport Properties Simple Diffusion Facilitated Diffusion Active Transport Solutes transported Thermodynamic properties Direction relative to electrochemical gradient Metabolic energy required Intrinsic directionality Kinetic properties Membrane protein required Saturation kinetics Competitive inhibition Small polar Small polar H20 glycerol H20 glycerol Small nonpolar Large polar Large polar 02 C02 glucose glucose Large nonpolar Ions Ions oils steroids Na K Ca2 Na K Ca2 Yes Table 83 7 Becker World of flag Cell 8277 eel Ham39m 62 al 2072 P65273072 7 77 Biology 7 ed Campbell cmd Reece 2005 P65273072 The NariK pump is an example of direct active transport ATP is expended to maintain high potassium and low sodium in the cytoplasm relative to outside the cell K and Na are pumped in opposite directions both against their electrochemical gradients Initial state pump open to inside E1 conformation 6 Two K expelled to the inside as the pump returns to initial state 6 K quot binding triggers dephosphorylation causing a conformational change back to E1 nib 2012 Pearson Education Inc INSIDE OF CELL 0 Two K from outside the cell bind to E2 42 I o39 c39 u Pump open to outside ready to start second half of cycle E2 conformation OUTSIDE OF CELL 0 Three Na from inside the cell bind to E1 rear i8 Na binding triggers phosphorylation ofa 1 subunits by ATP 9 A conformational change to E2 following 3 phosphorylation expels three Na to the outside of the cell The Na Glucose symporter is an example of indirect active transport Sodium is imported into the cell down its Initial stem oursms symporter open to outside OF CELL 0 Two sodium ions from outside the cell are bound 6 Release of glucose electrochemical gradient auows the empty symporter to return The decrease in free to initial state energy is used to pump glucose into the cell against a concentration gradient This enables accumulation of GIugse 9 Binding of sodium ions allows glucose binding and a subsequent conformational change 39 39 6 Loss of sodium high concentrations of ions 3 owed by glucose release to glucose in the cytoplasm inside relative to outside the cell 0 Sodium ions are released inside but are continually extruded to outside by a separate sodiumpotassium pump dashed line 9 Symporter opens to inside 2012 Pearson Education Inc Kinetics of competitive inhibition Illsi ES gt E P Competitive I SUbStrate inhibitor El 10 No inhibitor 3 I low 3 2 60 g I high 2 40 A competitive Inhibitor binds only to free enzyme 31 I medium I competes with S The inhibitor increases the 20 apparent Km but does not change Vmax o Substrate gt Biochemistry 7th ed Berg Tymoczko Stryer Note competitive inhibitors do not necessarily bind in the active site although they often do Kinetics of noncompetitive inhibition S El gtE5 gtEP l is l s product Substrate Noncompetitive inhibitor 100 NomthItor like Changing the amount of enzyme change in Vmax I low Relative rate I medium Substrate gt Biochemistry 7th ed Berg Tymoczko Stryer A noncompetitive inhibitor binds equally well to free enzyme and enzyme complexed with substrate I does not compete with S Consequently Vlmax cannot be attained even at high substrate concentrations l decreases the apparent Vlmax but does not change Km Methotrexate an anticancer drug that is a competitive inhibitor of dihydrofolate reductase O OH o M H i 395 O NH2 N N O WWW H2N N N o y f 25 9 O N 9 H s00105901 W H g 233 v augersz L l H DHF O a H2N N N H H Methotrexate MTX is a structural analog of dihydrofolate DHF a substrate for the enzyme dihydrofolate reductase that plays a central role in the bios nthesis y of purines and pyrimidines MTX binds DHFR 100fold more tightly than DHF MTX is used to treat some cancers and other conditions associated with DNA replication Competitive and noncompetitive inhibitors are examples of reversible inhibition Some inhibitors are irreversible egg by covalently modifying an enzyme Irreversible enzyme inhibition by the antibiotic penicillin Inhibition of growth of R Staphylococcus aureus by Penicilium mold OC RY 8 CH3 Hquot NH S CH3 0 fm PenicillinCOOH OH gt H Ser Glycopeptide Penicilloylenzyme complex transpeptidase enzymatically inactive Bacterial cell walls contain Damino acids that form crosslinks through the action of a glycopeptide trans eptidase Penicillin binds this enzyme and covalently modi ies it to abolish catalytic activity irreversibly Allosteric effectors bind at sites away from the active site and change enzyme activity Allosteric enzymes have two different conformations one that has high activity and one that has low activity for the substrate Effectors bind to an allosteric site or regulatory site Effectors regulate by binding and stabilizing one conformation Active Active Allosteric site vstlbstrate Allosteric sute Products site Slte Little or no A product AHOStCI 1C formation actlvator Highaffinity form Lowaffinity form of enzyme of enzyme Allosteric 0 Allosteric inhibitor activator Products Allosterlc 39 Little or no o o product 1nh1b1tor formation Lowaffinity form Highaffinity form of enzyme of enzyme 676 Bet 67quot World of 96 Cell 8 ed Hondim 62 52 2072 PedVJO Note allosteric inhibitors can be either competitive or noncompetitive Lipids and Membranes Lipids are hydrophobic or arnphipathic both hydrophobic and hydrophilic molecules that are soluble in organic solvents such as chloroform Fatty Adds steroids hydrophobic coo Fatty acnds amphlpathlc Saturated Unsaturated Palmitic acid AWNWCOOSO Oleic acid Stearic acid WC H Linoleic acidM lvvv COOH H O H H C 0 C l 0 H H C O C VVVW l Fourrin d h drocarbon R CH2 G39VCGTOI UNSHaded skeletonggf stzroids portion Phosphoglycerides more examples discussed in your reading Becker World of 96 Cell 857 ed Ham39m 62 62 2072 P65273072 Fatty Acids are long amphipathic molecules unbranched hydrocarbon Chain with a carboxyl double bond HEAD quotall W Wicijzjl 1 Tl fli ii1pl l Elm 1quot lf l l jjl t Lil a 2 H C 2 CH 2 All s1ngle bonds At least one double bond H20 g Relatively straight Kinked or bent shape CH2 g Packs together well Does not pack together well HquotC Iii CH2 V m H2C U CH2 0 co H26 VL CH2 32 7 sze 70 ed Sadam 62 al 2074 WH Freemm H2C CH3 Butter vs canola oil vs olive oil 327 Betear Will 0f 26 Cell 8 ed Hardin 62 al 20 72 P6473071 32 7 sze 70 ed Sadam 62 al 2074 WH Freeman Chains with cis double bonds have a strong kink in their structure Trans doesnt interrupt conformation as much as Cis easier to pack together RH Trans unsaturated fatty acids Cis Trans are rare in nature But are created synthetically in 9 various foods quotOff 70 353 39 39 4 4amp0de 8 9 Hog9 a Fair mm ammg z mm 6 I 0 0 6 I 9 9 Wyozz amz ed zz g arm s 5W6 H H Slde effect Of mead 2 0 elemz ea Complete chemical 1h d hydrogenation Partla Y ngCHath cholesz em eve5 52ml cm 6 H H H H H H 39 zmremea me of beam I I H rHHHHHH disease lllll II III HHHHHHHHHHHH IQI Double bond in the trans configuration hydrogenated doubled bonds became popular in WWII to make butter turn cis into saturated fat The Chemical properties of fatty acids Change their water solubility and melting temperature Water solubilitv Melting temperature 80 2 80 12 Largely 1nsoluble from talls g E 70 3 70 3 some solublllty from heads W 60 Less soluble with longer tails g 5 g 5 39 5 4o 2 4o 30 g 30 g 20 20 ItEu 10 g 10 0 v 0 quot 1o 1039 1o 12 14 16 18 20 g 20 1 2 3 5 Number of CiS Number of carbon atoms double bonds Unsaturated changing cis to trans double bonds increases melting temperature 7 J3 Beckeer LVord 0f 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 10 Phosphoglycerides or phospholipids have a hydrophilic head and two hydrophobic tails Hydrophilic head Hydrophobic tails Example phosphatidylcholine CH quotquot u E quotquotquotquotquotquotquot quot I 2 NCH33 Choline ChOllIIC 2 Phosphate Phosphate H 94 8 Glycerol Pg Glycerol E Phosphoglycerides often have one 2 unsaturated and one lt 9 saturated Chal Fatty acids 2 Fatty acids 94 0 Physical properties lt quot5 gt are affected by length I and S turatlon Of Fzg 573 Biology 7277 ed Campbell 51ml Reece 2005 P66175072 fatty acids like a cell Phospholipids can form lipid bilayers in water Water interiorand exterior Bilayer sheet Micelle hydroc 39 u molecules with o Bilayer phosph Phospholipid bilayer lt quotquot y 10 quot 39 574 Biology 7277 ed Campbell and Reece 2005 Panama Above the melting temperature Tm for a bilayer the membrane is a two dimensional uid In the uid phase lipid mobility is relatively high Within each bilayer lea et monolayer but quite low between lea ets liquid moving around solid locked in place The low intra lea et mobility makes it possible for cells to maintain different lipid compositions in the two monolayers membrane asymmetry Unaided 1 lipid ip event per month oz902199 279676 are enzymes fmz cam mung36 279239s reacz z39m zl39mzyey would require hydrophillic head to associate with phobic interior Why so slow 8 t 7 70 Beaeeer LVord 0f 96 Cell 8277 ed Ham39m 62 61 2072 P66175072 H 7 l I39 flipases lower the barrier critical bc synthesized in the cell and need to get in outer layer Lateral diffusion can be detected using uorescence recovery after photobleaching F RAP bleaching radiate strong enough light destroy Will fluorescence recover Diffuse need to be a liquid fluorescent molecules come in mobile g Laser Laser 8 i m d h o 2 I q o gt 339quot m C d H 5 Time Unlabeled Cell surface Laser beam Fluorescentlabeled Rate of diffusion of cell surface molecules labeled bleaches an area molecules diffuse fluorescence into bleached with fluorescent dye of the cell surface into bleached area area measured over time 7 7 7 Beteeer World of 96 Cell 8277 ed Ham39m 62 52 2072 Pearson You can read more about uorescence and uorescence microscopy in appendix A of your text Membrane uidity is in uenced by sterols that act as a uidity buffer Rigidity of Cholesterol decreases uidity above the melting temperature Hydrogen bond 0 J 39 Bulkiness of Cholesterol prevents lto phospholipids tails from packing together lowering the melting temperature Phospholipid lt Phospholipid f fggigwgg Cholesterol K vv vvv 7 75 B66 6rfv LVord of 196 C6ll 8277 66 Hardin 61 61 2072 P66175072 v Cell membranes need to be in the fluid state to function 7 75 B66 6rfv World of 166 C6ll 8277 6d Hardl 61 61 2072 P66175011 Membranes contain integral peripheral and lipidanchored proteins 1quot SURFACE quot 5 V 39 I c Q 0 I I I I s I I I I I I I I I I vv3939 I I I I I I I 39 b I I I Q K I I I I I I v 6 v Q I Q Q l I I I s 39 I Q Q I I I 39 I I I v I I I I o I I 9 I y I a I I 0 v I Q 1 v I 7 39 I I I 9 I I V 4 P I I Q I V v I I I v I I o P I I I I I v I l I P I I q 9 I v I i C V v Q V 39 I V C V 39 39 V 39 Q Y v V V i v 1 O 39 I I I I I I I I I I I I I I I I I I I I I r 39 I I I I I I I I39J quotquot I I I I I I I quot39 I I I I I I I I I I I I I I I I I I I I J I I I I I I I E E3 all I I I I I I I I I I I I IIEJIIIIIII 39 y g INNER cf 9 GP SURFACE quot quot KOJ anchor a Integral b Singlepass c Multipass d Multi e Peripheral f Fatty acid or monotopic protein protein subunit membrane isoprenyl protein protein protein anchor Y J Y Integral membrane proteins Lipidanchored membrane proteins 7 J9 Becker World of 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Transmembrane domains lipid b ayer quoti Iquot1 l 52 Single Multiple alpha alpha helix helices Fig 1017 Molecular Biology of the Cell 4th edition Protein segments spanning the bilayer generally have non polar residues in contact with the hydrophobic chains of the lipids An example of an alpha helical transmembrane domain EXTRACELLULAR 200 SPACE C E 1 2 m g 5 O 9 The hydrophobic core of mg W 3 39 398 blologlcal membranes are V m 9 generally 3 4 mm tthk Q 8 0 ms 393 me 39 ma 2 a m o g 39E i39 39o u C E m i CYTOSOL 21mm O nonpolar 1t The uid mosaic model of cell membranes a phospholipid bilayer in the uid phase with proteins dispersed throughout Lipids in each monolayer are in constant motion Proteins are also able to move laterally within the membrane though some are anchored to internal structural elements 39 Lipid rafts are membrane microdomains enriched in cholesterol sphingolipids and glycospingolipids more ordered than the surrounding 7 3 Biology 7277 ed Campbell 61720 Reece 2005 Pearmn Often contal l g many Slg ah g proteins Tertiary structure val Hydrophobic and van der Waals ser 1 quot interactions CH quot CH 2 H3C CH3 Polypeptide 0 H30 CH3 backbone H Hydrogen CH bond 1 O H Val Cvs CH2 CH2 Cvs CH2 Disulfide bridge Asp i Lys CH2 CH2 CH2 CH2 may 0 0 CH2 Asp Ionic bond Tertiary structure is the overall threedimensional shape of a polypeptide It arises from interactions between the side chains of the various amino acids The types of interactions involved include hydrogen bonds ionic bonds electrostatic interactions hydrophobic interactions van der Waals interactions and disulfide bridges Hexokinase domain 2 Quaternaw structure Quaternary structure is the overall protein structure that results from the association of two or more polypeptide subunits Term applies only to multimeric proteins Proteins made up of more than one polypeptide chain The interactions that determine tertiary structure also determine quaternary structure Heme Fig 311 Life 10th ed Sadava et al 2014 WH Freeman 2 quaternary structure of Hemoglobin To explore more structures of proteins and other macromolecules visit the Protein Data Bank httppdborgpdbhomehomedo Structure of Ribonuclease A httppdborgpdbexploreimoldostruotureld7RSAampbionumber1 Structure of Ribonuclease inhibitor complexed with Ribonuclease A httppdborgpdbexploreimoldostruotured1DFJampbionumber1 Substitution of one amino acid in the 3 subunit of hemoglobin is the cause of sickle cell disease Primary Normal hemoglobin Sickle cell hemoglobin structure 1234567 Secondary 3 3 b t 39 su um Zzgfg leasry j Exposed 1 h ydrophoblc 1 region Quaternary 0 Q 1 l3 5 structure Hemoglobin A Hemoglobin S 3 or Function q Tetramers do not Tetramers associate associate with each with each other 7 f other retain ability capacity to carry 39 q to carry 02 at high 02 severely capacity compromised Normal cells Fibers of abnormal Red blood are full of individual hemoglobin cell shape hemoglobin deform cell Into tetramers sickle shape 10 um The Perm httpwwwobsessedbybeautycom 201101mynewhairtoniguyIoose permreviewhtm Quaternary bonding interactions of the protein keratin can change hair curliness Disul de bridges Straigh hair Straigh hair reduce curl oxidize reduce i straighten 39oxidize gt gt Lehninger Principles of Biochemistry 6th ed Nelson and Cox 2013 W H Freeman Thermodynamics and Enzyme Kinetics I First Law of Thermodynamics Energy is neither created nor destroyed When energy is converted from one form to another the total energy before and after the conversion is the same you can t win Second Law of Thermodynamics When energy is converted from one form to another some of that energy becomes unavailable to do work you can t break even No energy transformation is 100 percent efficient perpetual motion machine Whenever a reaction occurs there is a net increase in the disorder entropy of the universe Entropy of a subsystem in the universe eg a reaction occurring in a test tube or a cell may increase or decrease but the overall entropy of the universe increases Less randomness More randomness less entropy more entropy Solid Liquid Gas httpwpsprenhacomwpsmediaobjects602616516Chapter17htm If entropy of the subsystem goes down order increases then heat Flows out of the subsystem and increases the entropy of the rest of The universe The change in free energy AG provides a means to follow the net change in entropy of the universe due to the reactions in the system in terms of quantities measured in the system see of matter call Molecular Biology of the Cell increased disorder uncreasod order Living matter has increased order at the expense of increased disorder outside the cell spontaneous reaction gt free energy deltaG goes does gt entropy increases The free energy of Y is greater than the tree energy of X Therefore ENERGETICALLY AG lt 039 896 quot 6 d39w39do39 Exe rgon IC FAVORABLE of the universe increases REACTK dunng the reactron If the reaction X bY occurred AG would gt h ENERGETICALLYt t o m 39 I UNFAVORABLE unwerse wou d become more REACTION mdmd Endergonlc Molecular Biology of the Cell ATP an important source of energy for many chemical reactions in the cell Exergonic reaction Hydrolyzed to ADP ATP hydrolysis H20 w ATP Adenine NH2 H C phosphate groupS c i v H OH OH Ribose J Y Adenosrne J Y AMP Adenosine monophosphate J Y Adenosine diphosphate J Y Adenosine triphosphate 89 Figure 65 uremssm Pi AG 73 kcalmol kcalmol unit of energy 1 kcalmol 7X1O3921J kinetic energy of a gas molecule at room temperature z 09 kcalmol Exergonic reaction ATP hydrolysis H20 Pi AG 73 kcalmol Endergonic reaction 0 O AG C NH4 0 34kcalmol 0 NH2 Glutamate Glutamine Net AG 39 kcalmol E 86 67 LIFE THE SCIENCE OF BIOLOGY Eighth Edition 2007 Sinauer Associates Inc and W H Freeman amp Co AG is a thermodynamic quantity it tells us about the direction of a reaction forward or reverse but tells us nothing about the speed or how a reaction occurs Free energy G Free energy G S substrate P product quotl quot AG 39 p Time Transition AGO is the AG of the reaction measured under quotstandard state conditions with pH 7 II Both of these reactions are exergonic or 1 spontaneous but the two reactions will not occur at the same rate Energy pro le of uncatalyzed and catalyzed reaction TranSition State lower the activation barrier A catalysis making reaction faster A613 uncatalyzed lowering free energy of Ea quot AC3 catalyzed T gt Substrate 3 Q C AC G for the 8 reaction L if Product Reaction progress gt Catalysts decrease the free energy of activation of the reaction AG stabilize the transition state 5 but not the overall magnitude of AG for the conversion of substrates to products The catalyst is not consumed in the reaction Enzymes are proteins that function as catalysts They are involved in virtually all cellular processes and affect many types of chemical reactions Reaction rate can increase by as much as 1017fold eg Orotidine 5 phosphate decarboxylase 78 million year reaction time reduced to 18 milliseconds Class Reaction Type Class Reaction Type 1 xidoreductases xidationreduction 4 I Yases Baum 0 group rcactiom lrom or addition ofa group to a molecule transfer of equot often with H with rearrangement of electrons forms double bonds 2 Transferases Transfer of functional groups from one molecule to another 5 Isomerases Movement ofa functional group within a molecule 3 Hydrolases Hydrolytic cleavage of one molecule into 6 Ligases Joining of two two molecules molecules to form a single molecule ase39 Table 61 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Examples of enzymecatalyzed reactions CHZOH CHZOH CHZOH H H H Sucrase o m9 gt CHZOH H OH OH H OH H Sucrose Glucose Fructose C12H22011 C6H1206 C6H1206 CHOH CH2OH CH 20H CH 2OH 0 0 O 0 H0 H H La eta 39 HO OH H H 0 0 H OH H H OH H OH H20 H OH H H HO OH H OH H OH H OH H OH H OH Lactose DGalactose DGlucose An enzyme catalytic cycle the basic principles 0 Substrates enter active site enzyme changes shape so its active site maximizes interaction with the substrates induced t d f SUBSTRATES S ENZYMESUBSTRATE COMPLEX ES 6 Active site is available for two new substrate molecules ENZYME E cquot Productsreleased 39 PRODUCTS P eSubstrates held in active site by weak interactions such as hydrogen bonds and ionic bonds Speci city for substrate 9 Active site and R groups of its amino acids can lower AG and speed up a reaction by acting as a template for substrate orientation stressing the substrates and stabilizing the transition state providing a favorable microenvironment participating directly in the catalytic reaction GSubstrates are converted into products The site on enzymes where substrates bind and chemistry occurs is the active site a Unfolded lysozyme b Folded lysozyme The active site contains Binding pocket Catalytic site May require something other than enzyme itself to work Cofacto rs Fig 62 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson o Coenzym es Prosthetic groups Rate of reaction Enzymes like many proteins are sensitive to heat pH and other environmental conditions Optimal temperature for Optimal temperature for pH optimum a typical human enzyme a typical enzyme of for trypsin thermophilic E pH optimum heattolerant s for pepsin bacteria g 6 0 5 o O 20 40 60 80 1 00 0 1 2 3 4 5 Temperature 00 What actors determine this SBIISiIilliW Chemist Protein SII IIGIIII G Fig 64 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Enzyme kinetics describes the quantitative aspects of enzyme catalysis and reaction rates 39 HOW FAST a reaction occurs not if the reaction Will occur Typically talk about initial reaction rates VO Enzymes only work if the reaction is already able to occur The if is determined by thermodynamics Reaction rates are in uenced by the environment and factors such as the concentrations of enzyme E substrates 8 products P and inhibitors X concentration of x I SUBSTRATES S ENZYMESUBSTRATE COMPLEX ES ENZYME E 39 PRODUCTS P The catalytic cycle can be modeled as a simple reaction SE ES PE Initial reaction rate Va is measured over a brief time at a particular E and initial 5 v0 is also called the initia Ill velocity Note your text uses v instead of V0 for initial velocity For early times the V0 ZIAP At MimiM concentration increases approximately linearly with time V0 is determined from this short time interval concentration 0 time httpwww Wiley comcollegepratt 0471393878studentanimations enzymekinetics At early times S has not yet decreased enough during experiment to affect the rate of P formation P has not yet increased enough for the reverse reaction P E significant ES to be gt Determination of initial velocity Vo of enzymecatalyzed reaction as a function of substrate concentration 5 Equilibrium T O 54 gt0 45 3 3 E 52 gt T D E 51 5 Substrate concentration S Time gt Can we understand the Initial rate V0 for each substrate Shape of this curve concentration is determined from the slopes of the curves at early time points MichaelisMenten Kinetics SEEST PE Neglect reverse reaction and assume S is constant since measuring initial velocity At steady state ES is constant V S max quot0 Km S Maud Menten and Leonor Michaelis httpwwwjbcorgcontent27949coverexpansion Michaelis L and Menten ML 1913 Die kinetik der invertinwirkung Biochim 2 42 333369 Glycolysis amp fermentation take place in the cytosol Under aerobic conditions the steps following glycolysis take place in the mitochondria for eukaryotes evolved from symbiotic bacteria within cell Mitochondria have two membranes The outer membrane contains porins Channel proteins with beta barrel structures that allow passage of molecules with molecular weights as large as 5000 Matrix Outer membrane Intermembrane space Inner membrane Cristae 703 13666673 World of 96 Cell 8277 ed Hardin 62 61 2072 P66173072 amino acids 100 molecular weight Under aerobic conditions most organisms can generate many more than two ATP per glucose 3 carbon compound into 2 carbon compound In the presence of oxygen pyruvate undergoes further oxidation to acetyl coenzyme A carbon relseased as 02 Acetyl CoA can be completely oxidized to C02 generating more than 30 ATP per glucose Via TCA cycle amp 39 Oxidatiye phosphorylation Cross mitochondrial outer and inner membranes Via porins and transporter respectively Coenzyme A o CytOSOI H a NAD Matnx of mitochuondrlon c o Aeroblc condltlons 02 From Lzo glycolysis l PDH I pyruvate dehydrogenase complex CH3 Pyruvate Acetyl CoA also generated from ox1datlon 98 Beaeeer LVord 0f 96 Cell 8277 ed Ham39m 62 61 2072 P66175072 Of fatty aCIdS fat 18 a source Of energY 39 The TCA cycle metabolizes acetyl COA releasing 2 C02 per turn and regenerating oxaloacetate acetyl COA 3NAD FAD ADP Pi gt 00CoA 2c02 3NADH FADHZ CoA SH ATP Acety39C A CoA 0000 W TCA cycle oxaloacetate Citrate Tricarboxylic acid cycle Citric acid cycle Krebs cycle THE TCA CYCLE 39 Oxidation Forms 3 NADH from NAD Forms 1 FADH2 from FAD Energy generation Forms 1 ATP Via GTP in animals 0000 Succinate ll 39ocoz 706 Bet 673 LVord of 96 Cell 8 ed Ham z 62 52 2072 P66273071 The TCA cycle accomplishes many things e V CoA sn Y H 0 go ll H o 3 l u H c c icx m H Pyruvate NAD 394 V THE 39I39GA CYCLE Enzymes l39hat catalyze These Reactions H c c o PDH Pyruvate dehydrogenase complex I V TCA1 Citrate synthase H c TCA2 Aoonitase l o TCAaz lsocitrate dehydrogenase TCA4 aketoglutarate dehydrogenase 0quot 39flc H TCA5 SuccinleoA synthetase 6 H TCAG Succinate dehydrogenase 39 TCAT Fumarata hydratase TCAe Malate dehydrogenase 39m39m 1 Two carbons enter the cycle as acetyl CoA which joins oxaloacetate to form 6C citrate 2 Decarboxylation occurs at two steps to balance the input of two carbons by releasing two C02 3 Oxidation occurs at four steps generating NADH in three steps and FADH2 in one 4 ATP is generated at one point with GTP as an intermediate in the case of animal cells 5 One turn of the cycle is completed as oxaloacetate is regenerated Each step in the TCA cycle is accomplished by a unique enzyme If you follow the carbons and the reactants products you can gure out many of the enzymes 708 Becker World of 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Various molecules can control the rate of glycolysis and the TCA cycle positive and negative regulation activation and inhibition of enzymes Why do some of these molecules act as activators and others as inhibitors in these athwa s Glycolysis v A1IVIP p y Glucose Acetyl CoA NADH ATP Enzymes That Catalyze These Reactions E1 PDH phosphatase w 1 pyruvate E2 PDH kinase a All other enzymes as in Figure 108 m e Fructose 6 phosphate Stl 11 ates Pymvate E1 Pymvme I m II dehydrogenase dehydrogenase 4 inactive active 55 F Phosphofructokmase i C I Fructose 16 bisphosphate lnh1b1ts Inhibits E2i T Citrate lsocitrate dehydrogenase NADH ADP Inhibition by ATP Citrate m are examples of w aketoglutarate L Oxidative L phosphorylation 7070 Beaeeer LVorlcl 0f 96 Cell 8277 eel Ham39m 62 al 2077 Bailquot52772222 Cummz39 gy 920 Biology 7 ed Campbell cmd Reece 2005 P66173072 Changes in free energy of the metabolic intermediates can be tracked PYRUVATE GLYCOLYSIS OXIDATION CITRIC ACID CYCLE 1OO 200 300 Acetyl 00A 1 400 500 Change in free energy AG in kcalmol 6OO 700 7 7 Lz 8 ed 5615161961 62 4 2007 WH Freeman All of the ATP made thus far has occurred by transfer of phosphoryl groups from a high energy phosphorylated substrate to ADP substrate level phosphorylation in the cytosol and mitochondrial matrix Mltochondrlon Substrate gt iiTriegeerijdgs 9 pvnuwms OXIDATION Hydrolysls Fatty acids oxldatlon 0 och0LvSIs CYTOSOL 4 I o ELECTRON TRANSPORT and PROTON PUMPING i But cells can generate much more ATP In using oxidative nnosnnorvlation Inner membrane Intermembrane space Outer membrane 9 ATP SYNTHESIS 707 Beteeer World of 96 Cell 8277 ed Ham39m 62 52 2072 Pearson In the presence of 2 cells generate ATP by respiration Cellular respiration includes glycolysis the TCA cycle and oxidative phosphorylation Electron shuttles span membrane MITOCHONDRION 39 6 NADH 2 FADH2 Glycolysis Oxidative gt 2 phosphorylation ED Glucose Pyruvate CC ton fans or and V Chemiosmosis CYTOSOL l 2 AT P 2 ATP about 32 or 34 ATP by substrate level by substrate level by OXidative phosphorylation depending phosphoryla on phosphorylation on which shuttle transports electrons from NADH in cytosol Y About Maximum per glucose 36 or 38 ATP 976 Biology 7 ed Campkel cmd R5666 2005 PedVJO The electron transport chain ETC or electron transport system conveys electrons from reduced coenzymes to oxygen NADH H 02 gt NAD H20 AGO kcall 11101 Large free energy release FADH2 oz gt FAD H20 AG 459 kcalmol Protons H are pumped from the mit matrix to the intermembrane space w red arrows rumquot W amp v t p Electrons are passed from one molecule to another yellow arrow Electron transport chain 975 Biology 7277 ed Campbell and Reece 2005 Penman Each complex has electron carriers that each have a standard reduction potential EO Succinate AE E E 2 O O acceptor O donor 50 02 10 c l I NADllolrozzxymeo f Complex II oxidoreductase succmzte39cgentzyme Q quot 3940 00 ox ore uc ase or NAID H dehydrogenase or succinate dehydrogenase complex 0398 E a measure of 1ts ml ex g g a x af nlty for 5 30 3 Complex III 9 g ClCthO S 39 Coenzyme Qcytochrome c 06 g quotg oxidoreductase E g or cytochrome blc1 complex g E gt t o 20 Tu 04 04 3 gr 2 Complex IV Cytochrome c 10 406 oxidase 02 0 F I e39 n O 08 Terminal electron acceptor forms water gt log lo 7075 Becker World of 96 Cell 8277 ed Ham m 62 61 2072 P66175072 number of electrons transferred 1n the react10n F is a constant Faraday s constant For each 6 pair from NADH 10 H are pumped from the mit matrix to the intermembrane space NAD H 2 2quotquot 2H Dehydrogenases MATRIX 2 2 Oxidizable substrates C3 2012 Pearson Education Inc 7076 Bet 673quot LVord 0f 26 Cell 8 ed Hardin 62 61 2072 Penman Electrons can also come from succinate Via FADHZ 39035 s quot w b a Q 39 TCA cycle Succinate dehydrogenase of succmm TCA cycle is actually embedded in the membrane as a quot Fumarate H component of Complex 11 211 gt39 j Coo pool x 39 2 m FAD FADH2 is a prosthetic 2H gt CoQ pool 4 2 W group of succinate dehydrogenase No Hl are pumped by Complex II Total of only 6 H pumped when electrons coming from TCA cycle s succinate thitht ti eil39 a oeuiiiy uuii v i u y 6 6 i n Inner membrane 7022 Beteeer LVord 0f Zae Cell 8277 ed Ham39m 62 61 2072 P65173072 1 u Outer membrane u lllllllllllllt ll 1 ll 1 l l l l l l l l l I l l I l l l l I l I l l l The crucial link between electron transport and ATP production is an electrochemical proton gradient Protons H are being pumped from the An electrochemical gradient is m1t matrix to the intermembrane space established where there are more A protons H in the intermembrane space than the mitochondrial matrix Proton motive force PMF n n 39 Vowq o r0t onii iquot O H W M u ATP s nthase uses the i v I COC OO J I39l gg39v quotquotW39 39lkw y I gtquot electrochemical gradient to 1 D form ATP ATP synthesis is coupled to electron transport 975 Biology 7277 ed Campbell and Reece 2005 Pearmn Chemlosmouc couphng ATP synthase FlFOATPase or F type ATPase has many components that work together to form ATP Remember Where did the H come from F0 F1 1 a 2 b 10 c subunits 1 5 3 OLB 1 y 1 8 subunits Static a H Channel GB ring making 3 catalytic s1tes makes 3 ATP per turn Static bs membrane anchor 5 immobilizes 0ch and connects to bs Mobile 39 l p 3 a y asymmetric rod or stalk CS39 rOtor that Spins as H ow paSt a 39 spins to activate catalytic sites on 03 one C forms ionic bond with a between arg on a and asp on C H entry protonates the asp rotates 110 of a turn per H viaA Net result per 10 H l C 8 part of the F1 stalk Mobile Free energy for ADP Pi to form 0116 1111 tum and 3 ATP ATP depends on local environment Energy of proton translocation produces a local environment favoring MATRIX 7079 Beteeer World of 96 Cell 8277 ed Hardin 62 52 2072 Pearson SY thCSlS Running the system in reverse produces a rotary rnotor Rotation of the c subunits direct observation 0 subunit rotation 22 micro meter actin lament axis atone end Mechanical Rotation of the c Subunit Oligomer in ATP Synthase FOFl Direct Observation Science 26 November 1999 vol 286 no 5445 1722 1724 The maximum yield of aerobic respiration is 38 ATPS per glucose for some prokaryotes and eukaryotes 39 The maximum ATP yield for oxidative phosphorylation from one glucose ONAD 10H ZFADH so2 34ADP 3er1 gt10NAD 2FAD 12H20 34ATP gt 10 X 10 Hl translocated gt 30 ATP 2 X 6 Hl translocated gt 4 ATP Energy yield for aerobic respiration Glucose 686 kcalmol ATP 10 kcalmol cell environ 10 X 38 380 kcalmol for ATPs 380 686 55 0o recovered Emrgyyied r rmem a m Gmore 68 6 6511 7770 ATP 70 Aral77ml 2 X 70 20 m moforATPr 20 686 3 recomrea Including the reactions of glycolysis and the TCA cycle give s 38ADP38 38ATP 602 6co2 6H20 Emrgy farawayfrom car e gz39 e 250o recomrea The actual yield of aerobic respiration is less than 38 ATPs per glucose Cyto sol Mit Oxidative Total 1 matrix phos yield Glycolysis 2 ATP 2 ATP 0 2 NADH gt 6 ATP 6 ATP lu Pyruvate NADH gt 3 ATP 6 ATP G l to acetyl 1 CoA Krebs ATP 2 ATP cycle 2 3 NADH gt 9 ATP 18 ATP X FADHZ gt 2 ATP 4 ATP In reality the value is lower PMF used for other things membranes leak complexes 1 and 111 can transfer a few electrons to 02 sometimes takes energy to transport NADH into matrix etc 6 Change in free energy AG in kcalmol GLYCOLYSIS PYRUVATE PROCESSING AND cnmc ACID cvcus ATPATP NADH x2 6x ATP 2 ATPX ATP X2 NADH x2 6x ATP Oxidative phosphorylation 6 I I L I I Ex c 01 co N go o o o o o 20 o o o o o 3 l l l l l Oxaloacetate In the absence of oxygen anaerobic conditions the TCA cycle and oxidative phosphorylation would both stop for humans and many other organisms colysis 1 NAD CoA r i 539 Ace 39 CoA w 39 7 r 139 l v 339 v c 39 row79 V g tuft v 3331 93 L u v 3 3 quot 6 quot390 9 I m 990 Mm 5 5 5 5 5 55 a a 9 39 39 39 J 7 V V 3 J 39 V I V CoA 77777777Wwarnrwnnnm 39 I i I a I i i I I I 39 QYQ P P quot F O QIIWA I p p o o mmmow 1 v u I 71k V p r VV My 4 K H R q l y 1r 1 39 14 I Q J r r A p I r 1 t 77 L Oxaloacetate 39 Fzg 975 Biology 7279 eel Campbell and Reece 2005 Pears T a FAD NAB Anaerobic conditions no 02 Succinate I Citrat 39 No terminal electron acceptor 0 CO2 No way to pass electrons from NADHFADH2 No way to generate proton gradient No way to do oxidatiye phosphorylation No FADNADregeneration Stops TCA cycle Starts fermentation But many prokaryotes can use alternatlve electron acceptors for respiration Less ef cient than 2 but more ef cient than fermentation Under normal conditions the electron transport chain is coupled to ATP synthesis why Uncouplers can abolish this coupling 39 Allows continued e transport and O2 consumption without ATP synthesis 1 r ughhl it V Iquotquot VquotI 39 I W39wu V ml crow H x g I 39 39r39 With an uncoupler l A 39 QQ V 39gquotquot 0 V A n a 5 A A L A A A l 0 3000000039 l H move down their gradient Lowers the proton gradient H don t pass through ATP synthase No oxidatiye phosphorylation i Fzg 975 Biology 7277 ed Campbell d l Reece 2005 Penman Uncouplers do not have to be channels Other drugs chemicals including poisons such as cyanide can affect the process in other ways Active transport is protein mediated movement against the electrochemical gradient moving solute against gradient ex Na ions 39 Couples unfavorable transport With ATP hydrolysis or some other source of energy such as favorable transport of another molecule Important for many cellular functions such as Uptake of essential nutrients Removal of wastes Maintenance of non equilibrium concentrations of certain iO S eg high potassium inside and low sodium outside Active transporters are also called pumps H sneaks in with 8 channel gt proton pumps get it out D1rect Ind1re ct H H H H H H H W H39l39 I OF CELL H Hquot Hquot w H s o 0 coccico o 01 o o o o o 0 ii 10 0 9 a o r to H o o quot0 9733 1 l 7 o o 0 o I I I 390 0 O 3 p 00391001 go 03 o 0 o 0 ii 0 o o 30 O O quotO 0 O O 0 If 0 I 0 0 a o o o o o o o o o v o o 1o 0 INSIDE OF CELL molecules gogng iii the same direction 673 sze 70277 ed Sadam 62 52 2074 WH Freeman Indirect transporters can be symporters or antiporters Simple diffusion facilitated diffusion and active transport move molecules across membranes in different ways Table 8l Comparison of Simple Diffusion Facilitated Diffusion and Active Transport Properties Simple Diffusion Facilitated Diffusion Active Transport Solutes transported Thermodynamic properties Direction relative to electrochemical gradient Metabolic energy required Intrinsic directionality Kinetic properties Membrane protein required Saturation kinetics Competitive inhibition Small polar Small polar H20 glycerol H20 glycerol Small nonpolar Large polar Large polar 02 C02 glucose glucose Large nonpolar Ions Ions oils steroids Na K Ca2 Na K Ca2 Yes Table 83 7 Becker World of flag Cell 8277 eel Ham39m 62 al 2072 P65273072 7 77 Biology 7 ed Campbell cmd Reece 2005 P65273072 The NaK pump is an example of direct active transport ATP is expended to maintain high potassium and low sodium in the cytoplasm relative to outside the cell K and Na are pumped in opposite directions both against their electrochemical gradients Initial state pump open to inside E1 conformation OUTSIDE OF CELL 6 Two K expelled to 0 Three Na from inside the cell bind to E1 5123 32229329 MW 60 3 i8 Na binding triggers phosphorylation ofa 1 subunits by ATP 6 K quot binding triggers dephosphorylation INSIDE OF CELL causmg a conformational change back to E1 42 m lt V7 W 2 I 9 A conformational 39 change to E2 following phosphorylation expels three Na to the outside 3 of the cell Pump open to outside ready to start second half of cycle E2 conformation nib 2012 Pearson Education Inc The Na Glucose symporter is an example of indirect active transport Sodium is imported into the cell down its Initial stem oursms symporter open to outside OF CELL 0 Two sodium ions from outside the cell are bound 6 Release of glucose electrochemical gradient auows the empty symporter to return The decrease in free to initial state energy is used to pump glucose into the cell against a concentration gradient This enables accumulation of GIugse 9 Binding of sodium ions allows glucose binding and a subsequent conformational change 39 39 6 Loss of sodium high concentrations of ions 3 owed by glucose release to glucose in the cytoplasm inside relative to outside the cell 0 Sodium ions are released inside but are continually extruded to outside by a separate sodiumpotassium pump dashed line 9 Symporter opens to inside 2012 Pearson Education Inc Metabolism is a complicated network of interrelated biochemical pathways in cells nabolic pathways synthesize cellular components atabolic pathways break down cellular components I 1h a PW Awn Mmua tuna u v u I quot quot 1 mm n mquot M new 7 39 u 394 39 4 m o i m 51 I 4quot i 39 l a Mil m39 h quotM Mquot ir V rrr u z 7 1 w wv w 1 nu quot u n 39V39K m Substrate Enzyme Product Complex network of interacting pathways w w m ImusAr mma gtnngtmltrov mmTrnuA nnNm nmrm n mnrunnmvnv L um mn rm mm nnv uan xnv LL Flucto wEP 39 up 39 quot quot LJneIup LMfwnlvl i LulrIuM uquot V m39 gt h l 1 u u r mw mme w wink ACT I I p I D a r o s v u 1 H k s I s zo gtogtmmo av r aU1roxvu011 oa oxnaIo I u 5 quot3 210 ua ozmdm 5 gm 5n 1 r We will mostly concentrate on a small but central part of metabolism glycolysis TCA cycle and oxidative phosphorylation 39 In I v we mm mm HM m t v9 v a 1 l w m I Wu u u I I w h m AW M m uquot Mn 39 mug J V Vrs 1 u u mma xgtxnngtmltron 5 w M N Central metabolism Imm04n1 mIMTrnMAnnNAI mmn n mnrucmu nv L Mm nmn 1m nlru nnv 39 39 y fmcto n tP y T mu 39Fu m LJ 1xnln m he 39 l quotN 0 2101 awn nu mw x 42ltvno o s v zodgtagtzamc av r 1quot m n o aroznucxu Oxidative phosphorylation tau 01310 avg 3121101 ua omnm Five broad stages To day Next lecture I wwel MI The glycolytic pathway O 2gt Fermentation O Cytosol 2 Pyruvate 1s ox1dized to generate acetyl CoA Acetyl CoA enters the tricarboxylic acid cycle TCA Mm matrix cycle where 1t 1s completely oX1dlzed to C02 Electron transport the transfer of electrons from reduced coenzymes to oxygen coupled to actlve Mit inner transport of protons across a membrane membrane The electrochemical proton gradient formed in step 4 is used to drive ATP synthesis oxidative Mm inner phosphorylation membrane and matrix For prokaryotes glycolysis fermentation and TCA cytoplasm electron transport chain oxidative phosphorylation plasma membrane The sugar glucose is the main energy source for most cells in humans and many other vertebrates but not necessarily for other organisms H O 1C H 2C OH 3 HO C H L 4 39 H C OH 5 H C OH 6 H C 39OH F23 22 7 Bea 673 LVord of 96 Cell 8 H ed Ham m 62 52 2072 P66273071 Glucose is a good source of energy because its oxidation is a highly exergonic process AGO39 686 kcalrnol c6leo6 602 gt 6co2 6HZO Glycolysis is a highly conserved process that converts one glucose molecule into two pyruvates reactions Glyl through GlylO glucose 2NAD 2ADP 2Pi gt Exergonic 2 pyruvate ZNADH 2H ZATP A G 20 kca1 mo1 a Phase 1 Preparation and b Phase 2 Oxidation and ATP c Phase 3 Pyruvate formation cleavage The sixcarbon glucose generation The two molecules and ATP generation The two molecule is phosphorylated twice of glyceraldehyde3phosphate are 3phosphoglycerate molecules by ATP and split to form two oxidized to 3phosphoglycerate Some are converted to pyruvate with molecules of glyceraldehyde3 of the energy from this oxidation is accompanying synthesis of two phosphate This requires an input conserved as two ATP and two NADH more ATP molecules of two ATP per glucose molecules are produced 000000 Glucose 000 2 Glyceraldehyde 2 3phospho 2 Pyruvate 3phosphate glycerate i golzpea wnEducamn39 39 c39 96 Bea 673 World of 96 Cell 8 ed Ham m 62 52 2072 P6473072 Ten individual steps make up the phases of glycolysis H H C O if Ii 39 H c H C H H c OH H C O I I I I Ho c H H C OH H C OH co co I II I I I HO C H HO c H Ho c H Ho c H Dihydroxyacetone H C OH H It OH H I OH H lt OH quot phosphate l J H I l Ho I 1 H C OH H C OH H C OH H C OH I I I H c OH H c o H C O H C O I I I I H co H H H H I Glucoses Fructose G H i OH phosphate phosphate bisphosphate H c o H Y Glyceraldehyde Phase 1 3phosphate NAD Enzymes that Catalyze These Reactions Gly1 Hexokinase GIy2 Phosphoglucoisomerase Gly3 Phosphofructokinase1 Sly4 Aldolase o GIyS Triose phosphate isomerase Gly6 Glyceraldehydeaphosphate c o 4 dehydrogenase Gly7 Phosphoglycerokinase H C OH GIyB Phosphoglyceromutase l GIy9 Enolase H O Gly10 Pyruvate kinase H 13bisphospho glycerate Phase3 II I I Th I H ceH QV HC 919 H 39 Phase 2lt Hl3 OH H c04 I I H H 2phospho 3phospho glycerate glycerate I H Phosphoenol pyruvate PEP Some tips for studying enzyme pathways Keep in mind the overall reaction starting and ending products Follow the reactants and products for each step Follow the functional groups andor molecular rearrangements Follow the number of carbons Follow the flow of energy and oxidationreduction when you can eg ATP NADH etc Think about the enzyme names 97 Bet 67quot World of 96 Cell 8 ed Hondim 62 52 2072 P66273071 Enzyme names can help you gure out the reactions they catalyze 39 Kinases catalyzes addition of a phosphate to a substrate Phosphatases catalyzes removal of a phosphate to a substrat reverse rxn of kinases 39 Carboxylases catalyzes addition of a carboxyl group to a substrate 39 Decarboxylases catalyzes removal of a carboxyl groups from a substrate 39 Oxidoreductases catalyzes an oxidation reduction reaction Hydrogenases catalyzes the reduction of a substrate add hydrogen Dehydrogenases catalyzes the oxidation of a substrate remove hydrogen 39 Isomerases catalyzes the structural rearrangement of a molecule Warning some BIIZVIIIGS are named for the reverse reaction which l0 also catalyze The rst phase of glycolysis requires 2 ATP molecules to split one glucose into two three carbon sugars glucose ZATP gt 2 glyceraldehyde3phosphate 2ADP T H c O T T T T 39 I t c 0 c H C H H C OH H C O I OH I 0 O l 0 Ho I H H C H C H I O I I I H HO c H HO C H HO c H HO c H D d t H I OH H I OH H cl OH O H I OH 39 l hrggzac eone 3 C sly1 Sly3 I Q H I I W I 9 H C OH H C OH H C OH H C OH I I I I H c OH H C O H c o H C 0 I I l H co H H H H H C 0H mm 6C Glucose6 6C Fructose6 6C Fructose16 6 C I phosphate phosphate bisphosphate H C O GIy1 Hexokinase H GIy2 Phosphogluconsomerase Gchra39dehyw 3C Gly3 Phosphotructokinase1 GIy4 Aldolase Sly5 Triose phosphate isomerase 3phosphate Hexokinase catalyzes the phosphorylation of gluco se HeXO monosaccha de O O H n Wlth 81X carbons C H Klnase enzyme that H C OH I catalyzes trans fer of quot0 f H quot0 H phosphate from ATP to quot I quot 0quot substrate H C OH I H I OH 39 6 lib905196522 10905065 074 H H 96 62 C Glucose6 phosphate The second phase of glycolysis generates NADH and ATP The second phase of glycolysis generates NADH and ATP Coenzyme NADH can be used to reduce other compounds carries reducing power NAD similarly acts as an oxidizer Other important redox coenzymes NADPHNADP FADH2 FAD B Oxidized form NAD 2 Reduced form lt9 H CONH2 I CONH2 H N N O P O Cm O O H H H H OH OH O NH2 N gt N N O P O CW O O H H H H OH OH 86 Figure 74 Part LIFETHESCIENCE OFBIOLOGY Eighth Edition 2007 SinauerAssociateanuand WiHiFreemanECo A NAD BH2 Oxidation Reduction Oxidation Reduction 86 Figure 7 4 Part 1 LIFE THE SCIENCE or BIOLOGY Eighth Edition 2007 Sinauer Associates Inc and w H Freeman 1 Co The second phase of glycolysis generates NADH 3c quot and ATP J Glyceraldehyde 3phosphate glyceraldehyde 3phosphate NADJr ADP Pi gt 3phosphoglycerate NADH H ATP Gly5 Gnyceraudehywamhosphate reaction by transferring electrons to an g39yce39ate dehydrogenase 39 sly7 Phosphoglycerokinase Charon acceptor like NAD o szk gagme z r d f d mmedfor z be LI 0 rename maxim Mai 22 5130 Munger 3 C H lt OH H cl o H NOTE there are two glyceraIdehyde3phosphates 332322 made per glucose V The third phase of glycolysis generates pyruvate and ATP 3phosphoglycerate ADP 3 3C C O Io H C H I Pymvate gt pyruvate I ATP 39 0 0 I H o H 3C H 3C c o c o I I II O H C OA H Clt OH I I quot c 319 H c OH H c 0 I I I H H H Phosphoenol 2phospho 3phospho pyruvate PEP glycerate glycerate Sly8 Phosphoglyceromutase GIy9 Enolase Sly10 Pyruvate kinase Note there are two pyruvate molecules made per glucose Also note other sugars can be oatabolized by the glycolytio pathway 2 net ATP and 2 NADH generated from glycolysis The accumulated reducing power must be used otherwise glycolysis cannot be sustained NAD must be regenerated from NADH V glucose ZNAD ZADP 2P1 2 pyruvate ZNADH 2H 2ATP Under anaerobic conditions no 2 fermentation regenerates NADJr from NADH by reducing pyruvate b Lactic acid fermentation Occurs in our muscles H 2pyruvate 2NADH 2H Zlactate 2NADJr during strenuous exertion From glycolysis In both cases most of the energy is still contained in the nal molecule No external electron acceptor C H 39 39 I No net ox1dation CH3 Acetaldehyde NAD 2 X ADP 2 X ATP H Enzymes that Catalyze These Reactions glucose ADH Alcohol dehydrogenase H C OH LDH Lactate dehydrogenase PDC Pyruvate decarboxylase CH3 PDH Pyruvate dehydrogenase 2 X Ethanol 2XNAD ZXNADH pyruvate c Ethanolic fermentation 2 pyruvate 2NADH 4 H gt 2 ethanol 2C02 2NAD 2 X end product lactate ethanol 98 Beaeeer LVord 0f 96 Cell 8277 ed Ham39m 62 61 2072 P66175072 Glycolysis amp fermentation take place in the cytosol Under aerobic conditions the steps following glycolysis take place in the mitochondria for eukaryotes Outer membrane Mitochondria have two membranes The outer membrane contains porins Nonspeci c channel proteins that allow passage of molecules with molecular weights as large as 5000 Matrix Intermembrane space Inner membrane Cristae 703 Beaearl Warcl 0f 96 Cell 8277 ed Ham m 62 61 2072 P66175072 Under aerobic conditions most organisms can generate many more than two ATP per glucose In the presence of oxygen pyruvate undergoes further oxidation to acetyl coenzyme A The acetyl group in acetyl CoA C 39t h d 39 l 39 39 IOSS ml QC on ma can be completely ox1dized to outer and inner membranes Via porins and transporter respectively C02 generating more than 30 ATP per glucose Via 39 TCA cycle amp Coenzyme A C toso j a NAD Matrix of mitochondrion 39 OXidative phosphorylation y c o Aerobic conditions 02 glycolysis I PDH pyruvate dehydrogenase complex CH3 Pyruvate Acetyl CoA also generated from ox1datlon 98 Beaeeer LVord 0f 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Of fatty aCIdS fat 18 a source Of energY 39 The TCA cycle metabolizes acetyl COA releasing 2 C02 per turn and regenerating oxaloacetate acetyl COA 3NAD FAD ADP Pi gt 00CoA 2c02 3NADH FADHZ CoA SH ATP Acety39C A CoA 0000 W TCA cycle oxaloacetate Citrate Tricarboxylic acid cycle Citric acid cycle Krebs cycle THE TCA CYCLE 39 Oxidation Forms 3 NADH from NAD Forms 1 FADH2 from FAD Energy generation Forms 1 ATP Via GTP in animals 0000 Succinate ll 39ocoz 706 Bet 673 LVord of 96 Cell 8 ed Ham z 62 52 2072 P66273071 The TCA cycle accomplishes many things e V CoA sn Y H 0 go ll H o 3 l u H c c icx m H Pyruvate NAD 394 V THE 39I39GA CYCLE Enzymes l39hat catalyze These Reactions H c c o PDH Pyruvate dehydrogenase complex I V TCA1 Citrate synthase H c TCA2 Aoonitase l o TCAaz lsocitrate dehydrogenase TCA4 aketoglutarate dehydrogenase 0quot 39flc H TCA5 SuccinleoA synthetase 6 H TCAG Succinate dehydrogenase 39 TCAT Fumarata hydratase TCAe Malate dehydrogenase 39m39m 1 Two carbons enter the cycle as acetyl CoA which joins oxaloacetate to form 6C citrate 2 Decarboxylation occurs at two steps to balance the input of two carbons by releasing two C02 3 Oxidation occurs at four steps generating NADH in three steps and FADH2 in one 4 ATP is generated at one point with GTP as an intermediate in the case of animal cells 5 One turn of the cycle is completed as oxaloacetate is regenerated Each step in the TCA cycle is accomplished by a unique enzyme If you follow the carbons and the reactants products you can gure out many of the enzymes 708 Becker World of 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Various molecules can control the rate of glycolysis and the TCA cycle Glucose Acetyl CoA NADH ATP Enzymes That Catalyze These Reactions G1 1 E1 PDH phosphatase YCO YSIS v B 1 2 PDH kinase All other enzymes as in Figure 108 Fructose phosphate Stlml ates pyruvate E Pymvate NADquot dehydrogenase 51 dehydrogenase F Phosphofructokinase macme z 2 acme Fructose 16 bisphosphate lnhiblts Inhibits CoA Malate NADH dehydrogenase Pyruvate Citrate Acetyl COA lsocitrate dehydrogenase NADH Q ADP NAD Inhibition by ATP and W Citrate are examples of NADH Succinyl CoA negative feedback L Oxidative phosphorylation 7070 3mm LVord of 296 Cell 8 ed Ham m 62 22 2077 3672227223972 Cummz39 gy 920 Biology 7 ed Campbell 472d Reece 2005 P62273072 Changes in free energy of the metabolic intermediates can be tracked PYRUVATE GLYCOLYSIS OXIDATION CITRIC ACID CYCLE 1OO 200 300 Acetyl 00A 1 400 500 Change in free energy AG in kcalmol 6OO 700 7 7 Lz 8 ed 5615161961 62 4 2007 WH Freeman All of the ATP made thus far has occurred by transfer of phosphoryl groups from a high energy phosphorylated substrate to ADP substrate level phosphorylation in the cytosol and mitochondrial matrix Mltochondrlon Substrate gt iiTriegeerijdgs 9 pvnuwms OXIDATION Hydrolysls Fatty acids oxldatlon 0 och0LvSIs CYTOSOL 4 I o ELECTRON TRANSPORT and PROTON PUMPING i But cells can generate much more ATP In using oxidative nnosnnorvlation Inner membrane Intermembrane space Outer membrane 9 ATP SYNTHESIS 707 Beteeer World of 96 Cell 8277 ed Ham39m 62 52 2072 Pearson Cytos keleton intermediate filaments dont need to know Actin white Microtubules DNA green Why are we devoting a lecture to cytoskeleto tracks for transport not fixed in live cell very dynamic support for cell kinesin and dynein motors dynein usually moves towards the minus end Enzymes that couple 2 CYCIGS kinesin usually moves towards the plus end Microtubule organization Chemical ATP binding ATP hydrolysis release ADP easy to see What is moving in and out neuron cell Mechanical body MT binding power strobe MT release binding causes release ADP triggers ATP to bind vesicle with bound dynein o vesicle with bound kinesin synapse microtubule A B Illil l l i l Figure 16104 Molecular Biology of the Cell 5e Garland Science 2008 has nothing to do with charge just a notation well defined organization in cell Microtubules have a polarity denoted plus and minus ends Minus ends are at the centrosome in the middle of the cell plus ends towards the outside Molecular motors carry cargo in either the plus kinesin or minus dynein direction Mechanochemical cycle of kinesin a Coiledcoil stalk ADP ADP Motor head Neck linkers M39crotUbule Qt I I Q Q Q 039 o39 039 o39 o 939 Q n I Forward motor binds Btubulin 9 releasing ADP 9 Q39Q39Q39 Forward head binds ATP er 4 3 Q Ar Conformational change in neck linker causes rear head to swing forward I Q Q 0 0 A F I r A A g ADP trailing head hydrolyzes ATP and w releases Pi Figure 1 822 Molecular Cell Biology Sixth Edition 2008 W H Freeman and Company b New forward head releases ADP bound bind to tubulin causes release of ADP comparison between molecular motor and racecar Dimension 15nm Energy ATP Velocity 2 MmSGC 133 lengthssec 310 mph Force 1 pN Efficiency 50 faster in terms of its own dimension tiny force in our terms twice as efficient 109 nm Gasoline 1O8 MmSGC 85 lengthssec 190 mph 1015 pN 2030 Microtubule structure how are they dynamic Electron micrograp 39 q amp l3 or tubulin heterodimer microtubule subunit this is a monomer technicall al habetatubulin dimer pro o lament single protofilament easy to break real MT more stable bonds in all dkec ons polymer N monomer ltgt polymer 50 nm n1 av A C microtubule Figure 1 79 Essential Cell Biology 3e Garland Science 2010 13 dimers around continually growing and shrinking from centrosome forms a stable structure they can always start from dynamic instability stop growing and shrink rapidly Microtubule assembly Lag phase Elongation Plateau nucleation phase phase 100 a plateaus bc co conc of free monomers depleting Q on Microtubule with E 3 subunits coming g E on and off 2 00 o V L a r 3 balanced 3 395 a Growing g e microtubule l 0 Individual Protofilaments dimers a g 09 moo I 0 99 go go Oligomers Time at 37 C gt 2012 Pearson Education Inc Becker s Figure 153 Monomers coming off are going oln Tubulin GTP ase Microtubule dynamic instability transitions between growing and shrinking I GTPbound 1 rapid growth with GTPcapped end GDPbound State at which growing or shrinking depends a I 33 gig a H on whether GD P or GTP bound 1 accidental loss of GTP cap I mo 1 rapid shrinkage I 4 1 regain of GTP cap 1 rapid growth with GTPcapped end 1 etc Figure 1616a Molecular Biology of the Cell 5e Garland Science 2008 Microtubule dynamic instability structural consequences of GTP hydrolysis GTPtubulin dimer I a B I exchangeable straight protofilament I GTP HYDROLYSIS CHANGES SUBUNIT CONFORMATION AND WEAKENS BOND IN THE POLYMER N curved protofilament I DEPOLYMERIZATION I I GDPGTP EXCHANGE p 4 Figure 1616b Molecular Biology of the Cell 5e Garland Science 2008 GDPtubulin dimer Microtubule dynamic instability differences between growing and shrinking microtubules 2179 TV N 39 1 quot r 3 r h 39 O E58173 Iquot quot o 39 quot quotvi 39 h quot 939 39 o 7 1 0 cj XEY39Q39 39 z quot 39 a 1 51239 s r 39 cap 24 C 3 3 quot v f 1 quotw 9 i to w w fab1 l g l I ffyv la 1 yr A r39 2 w e 39 13 tagquot I quot 5 1 M 3939 arr v 39 I O vb l v o s g 13 5 v 3 u xiij 39 K 1 pt 0 b 5 3 9 39E a d zquot 39 less stable nuts g g r o 0 o v w A 9 region of 3 n a o 9 mncrotubule 339 5 1 v 39x o39 t it 3 o o I 1 u a containing o 39 39 F as LM o o s quotI 39 GDPtubulln J gf a r fi 2 7 39 t1 dimers rsi 39 quot431 I 3 5 quot3 39 quot IV IIquot quot39 r I n I irfgf g 3 III I I 35quot o I 39p 7 Figure 1616c Molecular Biology of the Cell 5e Garland Science 2008 Microtubule dynamic instability in vivo T L y L I 7 39 l I v quot C V i D g a m l I 20 121 080 Lodish1802 capping unstable stable nucleus centrosome microtubule protein microtubules microtubules A i B I C Figure 1713 Essential Cell Biology 3e Garland Science 2010 The secretory pathway George Palade s pulsechase experiment quotgoing outquot pathway hormones enzymes membrane proteins follow secretory pathway but are not quotreleasedquot Copyright The McGrawHill Companies Inc Permission required for reproduction or display 6 THE DATA chose pancreatic gene as good modeI radioactive amino acids thOSG Will be secrete insulin and glucagon incorporated into any new proteins synthesized Time after chase quot First in the ER 5 miquot GOIQI Secretory vesicles Then seen in golgi 15 min Vesicles gt30 min then secreted 5 minutes after chase 6 Photo from Caro and Palade 1964 Journal of Cell Biology 29 p 479 g 3 The Rockefeller University Press chase change to incubation to non labeled amino acids see where the labeled amino acids go Brooker Figure 6 11 Location of ribosomes on rough ER membranes Rough endoplasmic reticulum Membranebound Free vs bound ribosomes ribosomes Free ribosomes That will be important for understanding how things enter the secretory pathway How do proteins get into the ER most likely bound ribosomes Ribosomes are on the outside Proteins need to get inside How do they cross the membrain s 39 f e 39 I 39 4 I a J l O a I 39 LM 39 39 ww r l39 quot i 39 s 9 39 S n I 39 39 M A 4 my w o sfof A t lign N c quot v 339 I a or t A vquot f 5 39 39 39 8 m39 1quot 39 quot39 Equot quotWe 39 39 g I 39 k d Rough ER u 05 um I lt16 2012 Pearson Education Inc Becker s Figure 4 15 ER microsomes How do newly synthesized proteins enter the ER purified from guinea pig liver with ribosomes attache mRNAs attached studded onto ER membrane Puromycin antibiotic drug stalls translation partially translate polypeptides rel gtased from ribosome prematurely 1 radioacti very slow to get anything int d400 200 1 000 800 600 400 200 or add puromycin here add puromycin o ribosome associated l l x This turned out to be helpful reductionist approach break something complex 1 into smaller understandable components Where are the radioactively labeled amino acids when puromycin is added 3 puromycin Inside or outside the ER 0 0 When released they go into ER i puromycin I Doesn39t have to be the fully translated protein I l product to get into the ER since these partial I proteins are getting in inside ER 4 8 12 16 20 24 Time mm Something at the Nterminus CM Redman DD Sabatini 1966 Vectorial discharge of peptides released by puromycin from attached ribosomes Proc Natl Acad Sci USA 56608 615 All free and bound ribosomes are the same only difference is mRNA being translated Those that make proteins to be imporariginal Signal hypotheSiS into the ER associate with the membrane Drew this largely correct schematic with very little information Those that make cytosolic proteins are Many secreted proteins don39t have a SUBUN T pom formymethione Nterminus change Polysomes o O Hypothesizesthatthereisa quotquot c oatmeweo FACTOR MEMBRANE signal sequence fully translated protein Biomembranes Vol 2 1971 pp 193 95 Ribosomemembrane interactions in eukaryot39ic cells G Blobel DD Sabat39ini Testing the signal hypothesis with a cellfree translation system Isolated ribosomes mRNA SUBUNIT POOL analyze proteins produced in this very simple system 0 Q Are these the same as those secreted from cells 5 9 n q 30 0 o 21991 quot c BlNDlN C3 FACTOR MEMBRANE Add ER microsomes add protease 1 2 l larger polypeptide separate proteins by molecular wei ht g smaller polypeptide twenty amino acrds are chopped off from the N terminus happening in the ER that39s what39s missing from the larger 1 translated in vitro from free ribosomes polypeptides from the free ribosomes 2 secreted protein Blobel G Dobberstein B Transfer of proteins across membranes l Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membranebound ribosomes of murine myeloma J Cell Biol 1975 Dec67383551 Testing the signal hypothesis with a cellfree translation system I I ER microsomes Protease secretory larger polypephde protein z quot5 smaller polypeptide cytoplasmic protein Blobel G Dobberstein B Transfer of proteins across membranes Reconstitution of functional rough microsomes from heterologous components J Cell Biol 1975 Dec673852 62 Revised signal hypothesis 1975 Part of the hypothesis is that there is a channel that lets the signal sequence through Signal peptidase cleaves the signal sequence Many difference sequences peptides recognized by signal peptidase codes for signal sequence 2 y signal sequence I fully translated cleaved signal sequence protein Binding factor recognizes signal sequence SRP ER signal sequences and SRP SRP receptor recognizes SRP and ribosome complex BINDING OF SRP 5 To SIGNAL SRPBOUND SRP AND SRP RECEPTOR PEPTIDE CAUSES RIBOSOME D39SPLACED AND RECYCLED A PAUSE IN ATTACHES To TRANSLATION SRP RECEPTOR IN ER MEMBRANE 339 TRANSLATION CONTINUES AND TRANSLOCATION BEGINS mRNA signal sequence on nascent polypeptide protein translocator SRP receptor protein in rough ER membrane Figure 1240 Molecular Biology of the Cell 5e Garland Science 2008 SRP Signal recognition particle recognizes and binds the Signal sequence and ribosome The SRP receptor in the ER membrane binds the SRPribosome complex The SRP receptor targets the whole complex to the translocator protein channel The newly synthesized protein passes through the translocator as it is synthesized Entry of soluble proteins into the secretory pathway 4 1 signal K peptidase cleaves signal from rest of the peptide inactive protein active translocator translocator mature soluble proteln In ER lumen Figure 1245 Molecular Biology of the Cell 5e Garland Science 2008 The signal sequence is cleaved by an enzyme called signal peptidase The newly synthesized protein is released in the ER lumen Integration of transmembrane proteins into the ER membrane hydrophobic section embeds into membr stops entering into the ER stays in the membrane in order to be tran I I elsewhere COOH starttransfer se uence q stoptransfer sequence A signal peptidase hydrophobic hydrophobic stoptransfer starttransfer peptide peptide binding site binding site mature transmembrane protein in ER membrane translocator protein Figure 1 246 Molecular Biology of the Cell 5e Garland Science 2008 Similar to the soluble protein except there is is a hydrophobic segment that acts as a stoptransfer signal Topological relationships between compartments lipid bilayer membrane protein lumenal contents Why are they all the same color as the outside of the cell why it is important to get into ER membrane plasma membrane lysosome Vesicular transport takes you from one to th crossing any membrane secretory budding vesicle vesrcle with contents selected for BUDD39NG I transport inner transport vesicle nuclear membrane in cytoplasm envelope outer FUSION l membrane Golgi endosome apparatus Figure 125 Molecular Biology of the Cell 5e Garland Science 2008 Figure 127 Molecular Biology of the Cell Se Garland Science 2008 Topological relationships ER only way to get into any of the vesicles lysosome enzymes need to go into ER lysosome plasma membrane rough ER secretory vesicle inner nuclear membrane envelope outer membrane Golgi endosome apparatus Figure 125 Molecular Biology of the Cell 5e Garland Science 2008 Roadmap of protein traf c ENDOPLASMIC RETICULUM I SECRETORY VESICLES LATE ENDOSOME KEY gated transport transmembrane transport EARLY ENDOSOME vesicular transport CELL EXTERIOR Figure 12 6 Molecular Biology of the Cell 5e Garland Science 2008 The chemistry of biology Course coordinator cjangsasupennedu Atoms Electronega vity Di erent types of bonds interactions Major functional groups in biology lVIark Goulian Macromolecules gouliansasupennedu 39 Sugars You should be familiar with the structures of atoms Life as we know it requires Atoms with the same 25 elements number of protons but Atoms are the smallest unit different numbers Of that retains the properties of neutrons are isotopes an element Atomic number number of protons Eectronquotquot oud Chemical symbol for helium Nucleus Protons plus neutrons Atomic wei ht Fig 22 Life 10th ed 9 Sadava et al 2014 WH Freeman Electrons Electronegativity X is the tendency to attract electrons complex concept Red major players in biology Elements differ in their electronegativities Electronegativity 1 07 4 18 2 13 14 15 16 17 L a Pauling scale a 1 No 10 15 29 Na Mg Al 51 Ar 09 12 3 4 5 6 7 15 16 K ca Sc 11 v 0 Mn Ga Ga 06 10 13 15 16 16 15 16 18 20 R0 Sr Y Z M1 Mo 11 In Sn SD 08 10 12 14 16 18 19 17 19 19 CS Ba La Ht Ta w Ho 11 Pb Bl Po Rn 07 09 11 13 15 17 19 18 19 19 20 Fr Ra AC R1 EX 59 811 113 Uuq 115 116 117 118 O 7 09 1 1 Ge Pr Nd Pm Sm EU Gd TD Dy Ho Er Tm YD LU 11 11 11 12 12 11 12 12 12 12 12 12 12 13 Pa U N PU Am Cm BK C1 ES Fm M0 N0 Lr 13 15 17 13 13 13 13 13 13 13 13 13 15 httpwww chemistryreference compdictabIeindex asp bond arbitrary definition of bonds and attractioninteraction The structure of molecules and materials comes from the atoms and the bondsinteractions that hold them together A X I difference in electronegativity between two atoms delta chi Nonpolar covalent bonds o AXltO5 39 l Equally shared electron Results in hydrophobicity one is more significantly electronegative than the other d I e ta Polar covalent bonds A X between 05 and 17 Unequally shared electrons COVALENT BONDS Results in permanent dipoles IONIC BONDS A X gt 17 Donated electron unlike charge attraction Van Der Waals Forces a class of weak attractive forces that result from dipole interactions These forces are present even for nonpolar molecules Fall off quickly with distance and weak even when close Induced dipole Induced dipole Induced dipole induced dipole interaction Induced dipole Even without charge electrons are fluctuating due to quantum mechanisms not in the same place impermanent dipole with movement of electrons Influences the movement of nearby molecules electrons chase other electrons away creating pos charge stick briefly How strong they are depends on details including the medium the molecules are in Proceedings of the National Academy of Sciences of the United States of America CURRENT ISSUE ARCHIVE NEWS amp MULTIMEDIA FOR AUTHORS ABOUT PNAS COLLECTED ARTICLES BROWSE BY TOPIC EARLY EDITION h gt Current Issue gt vol 99 no 19 gt Kellar Autumn 12252 12256 doi 101073pnas192252799 Th is lss ue CrossMark tlltk IO updatvs September 17 2002 Evidence for van der Waals adhesion in gecko setae vol 99 no 19 Table of Contents Kellar AutumnTi Metin Sitti Yiching A Lianng Anne M PeattieTl Wendy R HansenT Simon SponbergT Thomas W Kenny1I Ronald Fearing Jacob N lsraelachvili and Robert J Full l T httpwwwpnasorgcontent991912252full very flat surface Tokay gecko adhering to molecularly smooth hydrophobic GaAs semiconductor Why dont two objects stick together VdWs weak But also very few atoms get close enough together to activate VdWs only molecularly smooth surfaces huge issue in paint industry Hydrogen bonding another weak interaction Hydrogen bonds allow the partial positive charge 6 of an H in a molecule to be shared with the partial negative charge 6 of an electronegative atom nearby Hydrogen bonds play an important role in many of the remarkable properties of water crystal structure of ice electrical conductivity hydrophobicity 5 H rdo en shared with the ox en y g yg bonds Between two large molecules or Between two water molecules two parts of a large molecule Fig 211 Life 10th ed Sadava et al 2014 WH Freeman Nonpolar bonded atoms can experience hydrophobic interactions Nonpolar groupsmolecules move away from water for thermodynamic reasons Oil and water Hide hydrophobic regions from aqueous surroundings Often found in hydrocarbons molecules made entirely of hydrogen and carbon Amphiphilic or amphipathic molecules have both hydrophobic and hydrophilic regions fats fatty acids cholesterol soap Hydrophobic interactions are a major driving force controlling self assembly cell membranes protein structure Hydrophilic mix soap with water micelle structures g g iggggggggg ffg 5 p g bHayer httpcommons Wikimedia orgwikiFileiipidbilayerandmicellesvg m i ce I I e trying to minimize hydrophobic contact with water Hydrophilic Region httpcommons Wikimedia orgwiki FileCartoonofproteinhydrophobicin teractionjpg Bondsinteractions take different amounts of energy to come apart bond energies koaImol koaI6x1OA23 1000 Stronger bonds 4 c 3 s 858 C 00 Covalent bond 1 C H n 39 c C C e ergles 0 39 C N g s s U U 53 3 10 a c s a l39 Noncovalent bonds Hydrogen bonding and ionic Easier to break 1 Noncovalent Van der Waals and hydrOphobic 39 39 takes less E Vibrational thermal energy Weaker bonds 0391 Fig 22 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson r 2012 Pearson Education Inc There are a variety of functional groups in molecules formed by covalent bonds V0quot SIIIIIIIII IlIIIIW all 039 quot380 Iunctional QI IIIIIIS acidic have given up a proton extra proton i ll C 0 O P O39 NH3 0 Carboxyl Phosphate Amino a Negatively charged groups In neutral pH solutions b Positively charged group In neutral pH solutions 6 J 6 o 6 OH6 SH5 C C H ccc Hydroxyl Sulfhydryl Carbonyl Aldehyde Ketone c Neutral but polar groups Fig 25 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Typicaly made of carbons and hydrogen d Nonpolar amp uncharged groups Macromolecules are polymers or other molecular assemblies that act as cellular building blocks bacterial ions small cell molecules 4 phospholipids 2 DNA 1 30 chemicals RNA 6 g gt n o O 3 0 F1 7000 n roteins 1 5 c H20 P 0 Fl U1 polysaccharides 2 Fig 229 Biology of the Cell 5th ed Hardin et al 2008 Garland Science Proteins Nucleic acids DNA RNA Polysaccha rides Lipid assemblies such as lipid bilayers Polymers are made from covalently attaching monomers together polymerization How Short polymer Dehydration removes a water molecule forming a new bond Y Longer polymer K QH Unlinked monomer QH Unlinked monomer the polymer and monomer dont have same propertiesqualities depolymerization Long polymer one common form of CondensationDehydration polymerization reaction Hydrolysis adds a water molecule breaking a bond Fig 52 Biology 7 7 ed Campbell and Reece 2005 Pearson Shorter DOIVmer Unlinked monomer Hydrolysis Macromolecules and their monomers ar built from mall precursor monomer polymer Nucleoside P03 gt Nucleic acuds Phosphorylation tr39phosphates Nucleotides A quot2 NH gt Amino acids gt Proteins No 3 Amination 7 intermediaties 3911 gt was 02 l l OXIdatIon Glucose and other monosaccharldes gt Polysaccharides H20 002 Photosynthesis c 12 Pearson Education Inc Fig 215 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson Sugars have an aldehyde or ketone group plus two or more hydroxyl groups CmHZmOIn carbohydrate quothyd rates of carbon lt45 Fig 25 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson 0 Glucose is a monosaccharide 1 393 6CH20H H 2C 0H 3 HO C H 4 H C OH 5 H C OH 6 H C OH H a Fischer projection b Haworth projection 2012 Pearson Education Inc Fig 321 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson HI quot 2 F H cl 3 quotCH quotCH Aldosugar Ketosugar Fig 320 Becker s World of the Cell 8th ed Hardin et al 2012 Pearson disaccha rides monosaccharides H oc12 egCOSIdIC linkage CHZOH H a CHZOH H o H CHQOH CHZOH H O H 2 H O 0 H 2 1 CH20H 3 OH OH CHZOH H OH OH H H OH OH H aDGucose Fructose ocDGlucose Fructose What me 0 Sucrose manual made Ordina ry ta ble suga r this bond Examples of polysaccharides made from glucose monomers x 1 3 3 394 V quotLo H H i quot H H Cotton consists of cellulose with Starch is an example of a branched polymer over 6000 glucose monomers Regulation of HMGCoA reductase activity A AFTER REMOVAL OF B AFTER ADDITION OF LDL LIPOPROTEINS o Homozygote g 100 o LDL gt Z 3 r 39 None lt 3 A 3 E 39 Norma39 2 uglml 2 s g E 20 r U E D a m B C lt E C 0 Q 0 V 0 2 I 20 ugml 1 1 l 1 A g 1 O 8 16 24 32 0 4 8 12 Hours Hours Goldstein JL Brown MS Familial hypercholesterolemia identi cation of a defect in the regulation of 3hydroxy3methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol Proc Natl Acad Sci U S A 1973 Oct701028048 Binding and internalization of LDL BINDING 39gt INTERNALIZATION 02 H 1 P MA 0 0 l 50 100 50 100 LDL pgml LDL pgml U7 1 P H9 o39 0 Normal AM FH Homozygote Receptornegative Brown MS Goldstein JL Receptor mediated endocytosis insights from the lipoprotein receptor system Proc Natl Acad Sci U S A 1979 Jul7673330 7 Internalization and Degradation of LDL LA Normal Cells 12 a lntcrnalizcd 10 I Degraded D g 8 m 39 5 6 a I SurfaceBound 139 4 o 5 Brown MS Goldstein JL Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptorbound low density lipoprotein Cell 1976 Dec94 PT 266374 Visualization of LDL receptors by electron microscopy Anderson RG Brown MS Goldstein JL Role of the coated endocytic vesicle in the uptake of receptor bound low density lipoprotein in human broblasts Cell 1977 Mar103351 64 Endocytosis vesicles form from clathrincoated pits Figure 1348 Molecular Biology of the Cell 5e Garland Science 2008 Assembly of clathrin coat Budding Fission and vesicle release Internalization and Degradation of LDL LA Normal Cells 12 lnternalized E10 I Degraded 0 g 8 m 39 5 6 a SurfaceBound i 4 o 539 I g 25 o 0 l L l l l l I I I B 335 Ceus zslRADIOACTIVITY 10 b O SurfaceBound E A A lnternalized 3 8 l I Degraded v P O 1 D I 4 e 3 C 2quot AP 0 39d hI d I 0 10 20 30 40 50 60 120 Time at 37 minutes Brown MS Goldstein JL Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptorbound low density lipoprotein Cell 1976 Dec94 PT 266374 Membrane budding Saltquot r 9 vesicle clathrin V i 39 239quot K naked transport adaptor vesicle protein cargo adaptor protein receptor 0 cargo molecules M CYTOSOL Adaptor proteins mediate between membrane proteins cargo and coat proteins Bind receptors on the cytoplasmic side of the membrane Bind coat proteins which assemble to drive assembly of vesicles Binding and degradation of LDL T l B A 60 5 3 a t 0 9 H x DEGRADATlON 3 o i 3 f5 1 0 2 439 u 5 1 3 9 I J 8 o S Goldstein JL Brown MS Binding and degradation of low density lipoproteins by cultured human broblasts Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia J Biol Chem 1974 Aug 2524916515362 Receptormediated endocytosis of LDL lowdensity lipoprotein LDL LDL receptors Plasma membrane CYTOSOL M coated l u If pit 51 52 recycling endosome Q coatedx this part has u vesicle less SA but more We LDL particles gather free cholesterol enzymes lysosome Figure 1353 Molecular Biology of the Cell Sle Garland Science 2008 The entry strategy used by the influenza virus uillucnzu croosome VlrUS cellsurface v receptor hemagglutinin I HAI HA fusion reptiles now in 961 In um 1 unml mam imam pH change in the endosome a HOST CYTCISOL l viruses can exploit basic cellular machinery receptor mediated endocytosis virus has evolved to bind to those mechanisms Alberts Molecular Biology of the Cell Figure 2523 Polymers are made from covalently attaching monomers together H monomer Short polymer Unlinked monomer Dehydration removes a water k molecule forming a new bond I Long polymer Longer po ymer Hydrolysis adds a water Dehydration molecule breaking a bond I V Fig 52 Biology 7 7 ed Campbell and Reece 2005 Pearson Shorter DOIVmer Unlinked monomer Hydrolysis The 20 amino acids aa are the building blocks for proteins Carboxyl group a carbon H3N c H Amino IL group R groupor quotside chain different for each amino acid sidus inversus health probs messed up chirality Optical isomers enantiomers Mirror image very similar but distinct chiral objects fertilized egg started out symmetric develops into chiral human Life Sadava 8e t isomer Home 24 Mitch39squot Sin in C 1 W H 20396me c01er Glycine is the only amino acid that does not have a distinct D and L forms why Most proteins are composed solely of L amino acids glycine How does an organism use chirality of monomers to establish chirality in organism Cillia L isomers of amino acids Spiral shape cause fluid to circulate in a specific diretion All left handed amino acids The vocabulaw amino acid side chains Don39t need to know but worth making the effort Glycine Nonpolar Gly G I w H3N I r r r Ln r3 Methionine Met M SH polar CH3 CH3 CH3 CH3 CH ft o H 0 CH2 0 H3N c clt H3N c clt H3N c c I O l O l O H H H Alanine Valine Leucine Ala A Val V Leu L NH CI Iz CH2 H N I 0 I O 3 C C HN c c l o 3 o H H Phenylalanine Tryptophan Phe F Trp W CH3 l ICHZ H3C CH H3N c c O H Isoleucine lie I Proline Pro P The vocabulary amino acid side chains amine not protenated at pH 7 0H NH 0 C2 NH 5 70 OH H CH Polar I 0H CH3 I I I 2 CH CH CH CH CH2 CH I 2 O I 0 I 2 0 I 2 0 I O I 2 0 H3N C C H3N C C H3N I c H3N cc H3N C C H3N C C I o l o 0 l o I o I o H H H H H H Serine Threonine Cysteine Tyrosine Asparagine Glutamine Ser S Thr T Cys C Tyr Y Asn N Gln Q Acidic BaSIC NH NH NH O O 3 2 Q20 2 I I I C C IZHz C NH2 NH Electrically l CH2 0 CH2 CH2 NH CH2 0 charged l I I H3N C C CH2 0 CH2 H2 H3N C C I O I l I H3N C c CH2 0 H2 H 0 H I O l f H N c C CH H 3 I O I 2 O H H3N C C O H Aspartate Glutamate Lysine Arginine Histidine Asp D Glu E Lvs K Arg R His H You do not need to memorize amino acid structures and abbreviations but you will nd it is helpful to do so for more advanced biology courses reading research papers and following seminars You do need to be able to recognize properties based on the structures from the previous pages Table 32 Becker s World of the Cell 8th ed Hardin et al 2012 Pmrmason Education Inc Abbreviations for Amino Acids Amino Acid mail23 gril gfign Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Polypeptides as amino acid polymers Polypeptides Fl H 2 are polymers of amino acids 3N CH C C 39 A protein 0 consists of one or more polypeptides H204FgtH20 dehydration reaction 0 Amino acids are linked by peptide bonds Pentapeptide 0 NSerGIyPheAIaVaIC CH 3 H H2 c0039 Amino N terminus Carboxy C terminus Polypeptides as amino acid polymers R1 H R2 o Polypeptides I Where are the peptide bonds idS H3NCHiOH HNCHCOO How many aa residues are there What amino acids are they refer to a table What functional groups do you see What types of interactions can they 1ds undergo What is the net charge on this peptide Jolypeptides OH Pentapeptide NSerGIy PheAIaVaIC CHZOH H W H H Amino N terminus Carboxy C terminus Primaw structure A protein39s primary structure is simply the order of its amino acids amino acid residues usually written from amino end on the left to carboxyl end on the right by convention Part of the primary sequence of Human Pancreatic Ribonuclease A written with 3 letter abbreviations N terminus gt LysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerSer The entire primary sequence 124 amino acids written with one letter abbreviations KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLA DVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKH IIVACEGNPYVPVHFDASV If not specified assume N terminus is on left anad C terminus is on right If reversed completely diff protein Determination of the threedimensional structure of a protein structure by Xray diffraction challenge to get a protein to crystallize imperfections within protein defects Xray diffraction pattern obtained from the protein crystal diffracted beams beam of Xrays particle accelerator synchrotro radiation stop Xrays elecromagnetic radiation Protein Xray source crystal A C 1 B n as 0 3 s Rubisco K5 Figure 412 Essential Cell Biology Zle O 2004 Gartend Science Why Xrays and not visible light Not enough energy to excite electrons Need to be on scale of protein angstrum tenth of a nanometer typical wavelength of an xray in order to map reconstruct a protein Secondary structure A protein39s secondary structure describes how portions of its peptide chain are coiled or folded in a regular manner Secondary structure arises mostly from Hbonds between the substituents of the peptide bonds not the amino acid side chains Many weak bonds together are strong Regular shapes mainly alpha helices and beta sheets Portions of the peptide that form characteristic folds Mostly hydrogen bonds between peptide bonds Secondary structure ahelix coiled structure arising from Hbonding between every fourth peptide bond in the polypeptide chain quot f3 ai y l tv a r at p l I c 539 I f s L 5 39r39 39 n 3 o o 39 l r Side chains R groups Q 0 l3 I 9 F Peptide bonds Hydrogen bonds ain polypeptidr chains a a helix The ochelix is a righthanded helix Suppose all amino acids in the primary structure were changed to Damino acids Would the structure change Helices are chiral alpha usually right handed way to figure out thumb along plane if fingers going that that Ban 0 HQ Hbonds between every 4th amino acid 36 amino acids per turn with a displacement of 54 A Rgroups point outwards Secondaw structure pleated sheet structure arising from hydrogenbonding between the CO and N atoms in peptide bonds on different parts of the polypeptide chain for example in a hairpin or sheetlike structure The stretches of polypeptide may be in antiparallel as shown here or in parallel wwrw MAMWANA Disul de bridges or disul de bonds are covalent bonds between sulfur atoms in cysteine residues COOquot H3N Cysteine I oxidation CH2 2H 2equot lt Cystine 2H 2equot C CH2 reduction ystelne IIH NH3 IIH NH3 C00quot C00quot A chain GlyIleValGluGlnCysCysAlaSerValCysSerLeuTyrGlnLeuGluAsnTyrCysAsn 5 l 10 15 21 B chain f Phe ValAsnGln HisLeuCysGlySerHis LeuValGluAlaLeuTyrLeuValCysGlyGluArgGlyPhePhe TyrThrProLysAla 5 10 15 20 25 30 Porcine insulin Tertiary structure val Hydrophobic and van der Waals ser 1 quot interactions CH quot CH 2 H3C CH3 Polypeptide 0 H30 CH3 backbone H Hydrogen CH bond 1 O H Val Cvs CH2 CH2 Cvs CH2 Disulfide bridge Asp i Lys CH2 CH2 CH2 CH2 may 0 0 CH2 Asp Ionic bond Tertiary structure is the overall threedimensional shape of a polypeptide It arises from interactions between the side chains of the various amino acids The types of interactions involved include hydrogen bonds ionic bonds electrostatic interactions hydrophobic interactions van der Waals interactions and disulfide bridges Hexokinase domain 2 Quaternaw structure Quaternary structure is the overall protein structure that results from the association of two or more polypeptide subunits Term applies only to multimeric proteins Proteins made up of more than one polypeptide chain The interactions that determine tertiary structure also determine quaternary structure tetrameric structure Red oofaootor gt gives blood red color Heme has iron binds 02 Heme Fig 311 Life 10th ed Sadava et al 2014 WH Freeman quaternary structure of Hemoglobin 16 To explore more structures of proteins and other macromolecules visit the Protein Data Bank httppdborgpdbhomehomedo Structure of Ribonuclease A httppdborgpdbexploreimoldostruotureld7RSAampbionumber1 Structure of Ribonuclease inhibitor complexed with Ribonuclease A httppdborgpdbexploreimoldostruotured1DFJampbionumber1 paralell beta structure gt N and C terminilined up Substitution of one amino acid in the 3 subunit of hemoglobin is the cause of sickle cell disease lutamate ne char ed 9 g g Valine is hydrophobic bind together to Primary Normal hemoglobin Sickle cell hemoglobin hide from the water aggregate structure 1234567 Secondary V b t 39 t su unI 231555521 at quot 1 h ydrophoblc 1 region Quaternary 39 3 structure Hemoglobin 5 Function Tetramers do not Tetramers associate associate with each 39 with each other Q other retain ability capacity to carry 9 q to carry 02 at high 02 severely capacity compromised Normal cells Fibers of abnormal Red blood are full of individual hemoglobin cell shape hemoglobin deform cell Into tetramers sickle shape 10 Mm Intense pain cells bunch up and clog arteries The Perm httpwwwobsessedbybeautycom 201101mynewhairtoniguyIoose permreviewhtm Quaternary bonding interactions of the protein keratin can change hair curliness Disul de bridges Straigh hair Straigh hair reduce curl oxidize reduce i straighten 39oxidize gt gt Lehninger Principles of Biochemistry 6th ed Nelson and Cox 2013 W H Freeman EXPERIMENT M I I r39 U QUESTION Can simple molecules and kinetic energy lead to chemical evolution eX m e HYPOTHESIS If kinetic energy is added to a mix of simple molecules with high free energy reactions will occur that ng 193 Share produce more complex molecules perhaps including some with 00 bonds reproductiongrowth NULL HYPOTHESIS Chemical evolution will not occur even with Miller and Urey an input of energy respond to enVIronment EXPERIMENTAL SETUP Exp H2 NH3 cells f 39 W genetic material Hem metabolism Glass tubing T s k heat COOIIn9compartments contains dag 6 water vapor 9 poymers Large glass ask contains DNAgtRNAgtproteins Stopcock gases CH4 for taking quotquot3 quot2 What were the conditions like on early samples H H H 9 T HcH T lFCE earth H I water H H enser JL w t sources of energy lightning solar a er droplets radiation not most sophisticated S u I f V realiStiC eXp but firSt and flask gontains quotTl39ap bOllll39lg water Heat i9 idea was powerful r testing ideas PREDICTION Complex organic compounds will be found in the liquid water PREDICTION OF NULL HYPOTHESIS Only the starting molecules will be found in the liquid water RNA can act as genetic material but also RESULTS has a structural component catalyze 5i 0 Ho If we accept that you can get biologically Most popular idea RNA came first 5 OH HOE more advanced molecules from these Samplestaken fromthe qmd conditions how can we get to DNA water contain formaldehyde n89 hydrogen cyanide and several complex compounds with carbomarbon bonds Also these 3 components need each 39 di i t id m quotg 339quot quot 1f s other to get more which came first and CONCLUSION Chemical evolution occurs readily if Simple molecules with high free energy are exposed to a source of k kinetic energy 2011 Pearson Education Inc Freeman Biological Science Fig 31 Structure of a catalytic RNA Enzyme made of RNA rather than protein complex 3D structure w base pairing could catalyze replication of itself but still havent found one that does that 2011 Pearson Education Inc Freeman Biological Science Figure 411 Tertiary Structure of Tetrahymena Ribozyme RNA world model before the central dogma DNA is much more stable than RNA better at storing information 0 0 quotprebiotic soupquot Complex organic 5 molecules produced n RNA Proteins DNA by random chemistry RNA World RNP World LUCA Time gt RNP ribonucleoprotein LUCA last universal common ancestor BC this probs only happened once possible it was simultaneous but most likely once Cech TR The RNA worlds in context Cold Spring Harb Perspect Biol 2012 Jul 1 47 Building blocks for idea of compartmentalization b Fatty acid HO 0 c Carboxyl R group H2C CH2 H2C CH2 H20 CH2 HZC CH2 r Hydrocarbon HZC chain gtCH2 H26 CH2 H20 CH H C 2 Freeman 20113PearsnEduca oanc a 61 b A phospholipid Polar head cgfgggrroup hydrophilic Nonpolar tail hydrophobic Chemically simpler more likely started with fatty a a 3 E acid phospholipid L sphatgj Glycero 12 13 O O U B a s ll II micelle vesicle Freeman Figure 63 Exploring Life s Origins httpexploringoriginsorg Vesicle growth and division How to get growth and reporoduction Inc surface area without changing volume gt will become elongated G rowt h Agitate Repeated 1 c cles O Q Division y o o Q 03 Figure 3 Schematic diagram of coupled vesicle growth and division Reproduced from Zh u and Szostak 2009a and reprinted with permission from the Journal of the American Chemical Society 1 2009 growth and replication not stable splits into smaller vescles Schrum JP Zhu TF Szostak JW The origins of cellular life Cold Spring Harb Perspect Biol 2010 Sep 129 Model of a protocell Replication Growth and Division Figure 1 A simple protocell model based on a replicating vesicle for compartmentalization and a replicating genome to encode heritable information A complex environment provides lipids nucleotides capable of equilibrating across the membrane bilayer and sources ofenergy left which leads to subsequent replication of the genetic material and growth of the protocell middle and nally protocellular division through physical and chemical processes right Reproduced from Mansy et al 2008 and reprinted with permission from Nature Publishing 132008 Schrum JP Zhu TF Szostak JW The origins of cellular life Cold Spring Harb Perspect Biol 2010 Sep 129 Cells are the fundamental unit of life Cells are both distinct entities and building blocks of more complex organisms Cells created from preexisting cells by division Cells contain heritable material which is maintained over division First theory had 3 facets first 2 were same but 3 was Cells form spontaneously we think this is wrong now All living organisms on this planet are descended by an unbroken series of divisions to an ancestral cell that came into being over a billion years ago Kim Nasmyth Early cells Look very similar structurally much simpler Copyright The McGrawHill Companies Inc Permission required for reproduction or display r 3 a Fossil prokaryote b Modern cyanobacteria a Stanley M AwramikBiological Photo Service b Michael AbbeyVisuals Unlimited 35 billion years old Modern cyanobacterium almost certainly first life Prokaryotic cells E coli electron micrograph Generic prokaryotic cell schematic Plasma DNA cell wall flagellum membrane rI bosomes Figure 118a Molecular Biology of the Cell Se Garland Science 2008 Can photosynthesize huge diversity know very little about so much of them Figure 118b Molecular Biology Kenya Garland Science 2008 very small The three major domains of life 2 classes Bacteria we know a lot about the pathogens diverse environments pH temp chemicals what looks alike to us is really very similar in genetic diver ity terms Z gl 4 Halo ferax 3 x 39 Aeropyrum l 39 cyanobacteria aParameCium Methanothermobacter Bacillus I Sulfolobus Methanococcus Dictyoste um l I 4 E coli f Euglena u Trypanosoma 39 Thermotoga common I I Grardra ancesmquot 1 change1O nucleotides Trichomonas Aqurfex cell Figure 121 Molecular Biology of the Cell 5e Garland Science 2008 scale Major features of eucaryotic cells structural specialists while prokaryotes are genetic specialists microtubule centrosome with 39 extracellular matrix pair of centrloles chromatin DNA nuclear pore 7 nucleolus peroxsome f db d ribosomes r n n eea 0U E39 kymso39 Golgi Intermediate plasma nucleus endoplasmic mitochondrlon apparatus laments membrane reticulum Figure 130 Molecular Biology of the Cell Se Garland Science 2008 The drawing depicts a typical animal cell but almost all the same components are found in other eucaryotic cells Origins of eukaryotic cells PROCESS ORIGIN OF THE NUCLEAR ENVELOPE 1 Ancestor of eukaryotes 2 Infoldings of membrane rearranged to get dif strL prokaryotes do this to inlt 3 Eukaryotic cell Nucleus Endoplasmic reticulum How could we have gotten to eukaryotes Need a nucleus Reason this is an attractive idea this is what a nuclear envelope looks like Also ER is an extension of the nuclear membrane Advantageous evolutionarily bc protection of genetic material and keep things out of nuclear envelope and bring ctltmgs in when needed transcription factors regulate 3 Sdoncentrations of things within and without Compartmentalization can modify RNA before translation no ribosomes in nucleus dont want to be spliciing RNA as transcribing Evidence Membrane infoldings in bacteria Nuclear envelope and ER membrane structure L 2011 Pearson Education Inc Freeman Biological Science Figure 298 A Hypothesis for the Origin of the Nuclear Envelope Origins of eukaryotic cells PROCESS THE ENDOSYMBIOSIS THEORY Highpotentialenergy 0 Electron transport High ATP carbon compounds 2 chain 7 yield Aerobic bacterium Anaerobic eukaryote 1 Eukaryotic cell engulfs bacterium 2 Bacterium survives 3 Endosymbiosis Both benefit 2011 Pearson Education Inc Freeman Biological Science Figure 299 Proposed Initial Steps in the Evolution of the Mitochondrion Anaerobic not respiringusing 02 Evidence Mitochondrial size and replication Mitochondrial ribosomes Mitochondrial double membranes Mitochondrial genomes different and separate from DNA of host cell Ground up cells Filtrate can t have cells inject cellfree filtrate into another chicken which would develop a tumor Transformation cells in the second chicken dividing in uncontrollable way What can you guess aboI If what is causing the transformation enzymes Chemical signal DNA RNA Virus Virus can pass through filter infectious Rous sarca39n RSV DNA RVS RNA genome Reverse transcriptase as violating the central dogma Question if virus can transform cells what genes are causing the cells to proliferate l Hybridize W RSV RNA 1 Reverse transcribe thSV RNA Virus replicates and transforms WT Mutant can also replicate but not transform viralg 10 ei sm2lller SARC gene stands for sarcoma allows for transformation How did they figure out what this gene is l HAP column Mrr I sarc DNA wt viral genome td transformation defective isolate DNA fragements by chromatography SARC is not specific to the virus all humans have it virus is slightly modified form can lead to scary cancer Why does viral gene lead to tumors SARC codes for a protein kinase enzyme that catalyzes addition of a phosphate group to another protein phosphate from ATP viral SARC activity of kinase not controlled in a normal way kinases control lots of different aspects of cellular behavior slight changes BIG deal Retroviruses and oncogenes Nobel Prize lecture 1989 Harold Varmus ATP and GTP just substitue guanine KINASES ATP Adenine NH2 OH OH Ribose Y J Adenosine x J Y AMP Adenosine monophosphate x J Y Adenosine diphosphate k Y J Adenosine triphosphate 89 Figure 65 LIFE THE GTP guanosine triphosphate substitute guanine for adenine ATP hydrolysis reeases ENERGY ATP stores energy phosphoanhydride bonds ATP inorganic phosphate Pi Figure 257 Molecular Biology of the Cell Sle 0 Garland Science 2008 The hydrolysis of ATP to ADP and inorganic phosphate Kinases we care about what happens to phosphate not what energy is used for Protein Phosphorylation can have huge effects covalent modification OH serine I side chain CH2 pROTEIN KINASE PROTEIN PHOSPHATASE Figure 3 64a Molecular Biology of the Cell 5e GD Garland Science 2008 A protein kinase catalyzes the transfer of a phosphate group from ATP to an amino acid side chain of the target protein The phosphate is added to the OH group of a serine side chain as in this example or a threonine or a tyrosine Removal of the phosphate group is catalyzed by a protein phosphatase seemingly negligible mass why do we care pea on top of a washing machine What does this phosphate group do 2 optiosn adding a negative charge change electrostatics of protein refolding differently disrupts hydrophobic causes it to become recognized by another protein whereas it wasnt recognized before signalling molecules as important as kinases Human bladder carcinoma Oncogene discovered the Ras gene oncogene responsible for transformation Ras sarcoma very similar to genes all our cells have with cancer nrngene founding member of group called GTPases removal of phosphate from GTP like kinases but GTP seem similar but act differently in this case we don39t care what happens to the Recipin phosphate mouse cells Oncogene we care what happens to GTPase Itself mm mm can be in inactive or active phase bladder depends on if its arrinnnm l m Informed mome rel Is Figure 1522 The Cell A Molecular Approach 2nd edition Cooper GM GTPbinding proteins as molecular switches hydrolysis switches it off release of GDP switches it on regulated by other proteins can be regulated by cell Pi GTP SLOW FAST HYDROLYSIS 9 l I T STA l7 l w faquot I Hy J r w w l r Figure 371 Molecular Biology of the Cell Se Garland Science 2008 depends on if its bound to GTP or GDP Examples of cellcell communication Signaling more generally Chemical signals electrical signals light mechanical signals Today focus on chemical signals Copyright The McGraw Hill Companies Inc Permission required for reproduction or display Membranebound signaling molecule A a Direct intercellular signaling b Contactdependent signaling c Autocrine signaling a Cell signal to themselves 3 and to nearby cells Endocnne cell d Paracrine signaling 6 Endocrine Signaling o 00939 9Q 9 399 a C9 S39gnal to nearby cells of Cell release Signals hormones that travel long distances to affect other cells Brooker Figure 93 Membranepermeable vs impermeable ligands PROCESS LIPIDSOLUBLE SIGNALS ARE PROCESSED DIRECTLY pIasma rsmroid 1 Arrival of signal membrane hormone 39 g V vwmmmwmwmmmum 39 g w m gmmWW minimummmwwmmlimIIIIIIII IIIIIgIgiIWm l um r www l ilygm a a 2 Signal reception Receptor in cytosol WIINIIIIWWlWN g w mm m Q l m Wm Willilllilii williilm I 39quotWWImiliil l wlwlrmWm m 3 Direct signal response 2011 Pearson Education Inc Figure 813 PROCESS LIPIDINSOLUBLE SIGNALS REQUIRE TRANSDUCTION Q lntercellular signal Receptor PrOtein 1 Signal reception in membrane A 39 mm will Ill Jlll llHlWllllllllllllUlNllllH 39 Num 11le r umm 39 Ml l s11uummm mltwuwmmlllIn will Willlwnunlmmmm mp H WWW mmummmw IUlllrIl39Jllllllllm l 39 n1u w lh m113911l l m NW WW Wm Intracellmar 2 igna39l signal 4 V transduction i Q 3 Signal 1 amplification 1 39 willllllll lllllIIWIllllllwWNW WW W M pl 5 wmwwmWmmy l l lll mgnwuwmm MUN a IMWIRW UMWIlllWIMIlI lM quotI 3IInIImIIIIIWQIW ligMHnImminnowm IUIlUlluw mu 1le nnynmmm MInu n gig1 blu will New quot o 39 I 39 c a c on o q 3 S a a 0 III 39 In A o g o 39 quotaquot c M Mgf I 39 2011 Pearson Education Inc Figure 814 Typical receptor tyrosine kinase pathway signal molecule EXTRACELLULAR M SPACE inactive Ras protein activated Ras protein CYTOSOL activated RTK adaptor protein Rasactivating protein Figure 1631 Essential Cell Biology 3e Garland Science 2010 Ras activates a MAPkinase signaling module plasma membrane m CYTOSOL GTP activated Ras protein l l l l protein X protein Y an 39iP ion z h al39ISGiP on regulatorA y regulatorB l I I II II I I 39 I Figure 1632 Essential Cell Biology 3e Garland Science 2010 dProkaryotes Central dogma Prokaryotes Transcription and Translation occur in cell body goes to ribosom 9 translated v protein 39Il39anscription DNA is transcribed into an RNA copy 10lyp pt39de Translation Ribosome mRNA is translated into a polypeptide at the ribosome a Molecular gene expression in prokaryotes Transcription Produces an RNA copy transcript of a gene Most genes produce messenger RNA mRNA that specifies the amino acid sequence of a polypeptide Translation Process of synthesizing specific polypeptide on a ribosome Central dogma Eukaryotes Transcription RNA processing where pre mRNA is processed into functionally active mRNA Translation Eukaryotes Transcription occurs in the nucleus Innovations DNA in nucleus genes to mRNA PremRNA in nucleus 9 splicing 20000 genes in human genomes flies have 16000 999 can be spliced in an alternative way to make family of related proteins 7x20000 polypeptides Translation occurs in cytosol J Q r iiquot uquot quot A g c g re ernR X Transcnptlon if r 39 4 l a n 3939 G HH 39 39 1 7 Entry into cytosol 1 Translation RNA processing l vgrPolypeptide gRibosome b Molecular gene expression in eukaryotes Central Dogma Interesting eukaryotic innovations Many genes code for RNA molecules that do not function as mRNAs and are not translated into proteins 0 These RNAs perform important functions in the cell Structural Regulatory Information can flow in the opposite direction from RNA back to DNA 0 For example some viral genes are composed of RNA and use reverse transcriptase 5 gt3 RNAdependent DNA polymerase a viral polymerase to synthesize a DNA version of the RNA genome Single genes may encode many different but related proteins by producing sets of related mRNAs by alternative splicing tRNA 9one of the first RNAs discovered 9 structural bind to amino acids and participate in translation Also Small families of small RNA molecules that regulate other RNAs stability of mRNA Ribosomal RNAs form structure of ribosomes some Functional classes of RNA RNA Function B A E Size n messenger mRNA protein coding variable small nuclear snRNA splicing 80350 transfer tRNA translation 7580 ribosomal rRNA structural amp 238 2904 1 enzymatic 1 telomerase TERC structural 451 2 micro miRNA regulatory 21 27 long noncoding lncRNA regulatory Xist gt17kl mRNA can be small or large Form machinery of translation transfer and ribosomal 2 types of ribosomal 23s and 16s ibosomal catalyzing the polymerization of amino acids into polypeptides 39 39 Making a peptide bond is catalyzed by the large ribosomal RNA B BaCterla CO In eukaryotes the ends of linear chromosomes have to be A ArChaea hU man repaireEnzyme telomerase 2 polypeptide RNA E E U ka rya Micro eukaryotes 1000 of these regulating Whole chromosomes Turning off expression of one X chromosome in animals Central Dogma Interesting innovation Information can flow in the opposite direction from RNA back to DNA 0 For example some viral genes are composed of RNA and use reverse transcriptase 5 gt3 RNAdependent DNA polymerase a viral polymerase to synthesize a DNA version of the RNA genome HIV Viral particle 2 chromosomes identicalHdlMIREplica tiOn Genome RNA single stranded Polarities on nucleic acids Copies first into single stranded DNA then into double stranded DNA Removes RNA half then copy into DNA Catalyzed by reverse transcriptase Double stranded DNA integrates into genome Uses host RNA polymerase 5 to 3 JV g V 39 Vt 0 3 o 3 00 2 5 a mo Zio ofquot SSRNA MAL l IADNA l dsDN 2322 gt1 Viral polymerases Virus Polymerase gene Activity Endo Ribonuclease H activity degrade RNA High error rate no proofreading activity 10 kb 10000 base pairs one in ten genomes mutate Coming soon Monday 32 HIVAIDS History cures and Penn Robert Doms The Children s Hospital of Philadelphia Perelman School of Medicine at the University of Pennsylvania Viruses Virus Very small infectious particle constructed from nucleic acid and protein and sometimes lipid that can reproduce only in living cells Readingviewing Required httpenwikipediaorgwikiViralreplication Required introductiontovirologyOt pdf Required httpwwwvoutubecomwatchveStGODinO8w Interested in Virology httpviralzoneexpasyorg Viruses come in many shapes In uenza Vitus Adenovims 100nm 90nm G e Papillomavirus Hepatitis C virus Hepatitis B virus Hepatitis A Vitus Pawoviws Ebohvims SOnm Dengue Vitus 42nm Poliovirus 20nm 80x9700m SOnm 30am Don t do anything on their own need a cell good probes into mechanisms of cell activity Viral classification based on genome structure and replication strategy 1 Class Genome Examples 1 dsDNA adenoviruses poxviruses 2 ssDNA Bacteriophage Ext 74 3 dsRNA Reoviruses Rotavirus 4 SSRNA strand Rhinovirus Yellow Fever virus Poliovirus 5 ssRNA strand Influenza Measles virus Ebola virus 6 ssRNA strand Retroviruses HIV1 Mouse mammary empey DNA intermediate tumour virus Murine leukemia virus 7 dsDNA hepatitis B virus employ ssRNA intermediate Genome structure characterized by some quotrl i GddEaittPn39 esaletglig i39l 93 some 3339 single stranded Slngle Strange Jr positive polarity function as mRNA Posrtive polarity RNA looks like mRNA negative polarity complementary to mRNANeQat39Ve DOIar39ty the COmPIGment Of that need RNA dependent RNA polymerase Viral genome mRNA is the complement ds double stranded Viral classification based on genome structure and replication strategy 1 hlyyuguuuqzx J E Q 39 l lit 1 h q cocuyygutn39tnr tng l ib C yaquot I Genetic material present in the virion Groupl Group II Group III Group IV GroupV Group VI Group VII DNAI DNA RNA I RNA RNA RNA DNMH L Revlon3e trans dp on DNAl H new transcription 39 39 mRNA ds double stranded 1 David Baltimore classification system ss single stranded positive polarity function as mRNA negative polarity complementary to mRNA proteins Viral polymerases Virus Polymerase gene Activity 5 gt3 RNAdependent DNA polymerase HIV Reverse viral Endo Ribonuclease H activity transoriptase 5 gt3 DNAdependent DNA polymerase error rate 1 x 10395 errornuoreplioation 5 gt3 RNAdependent RNA polymerase Influenza RNA ol merase viral p y error rate3x10394errornuoreplioation Favipiravir T705 Avigan Toyama Chemical High mutation rate Made to inhibit RNA polymerase under investigation to stop Ebola Viral classification based on genome structure and replication strategy 1 Class Genome Examples 1 dsDNA adenoviruses poxviruses 2 ssDNA Bacteriophage Ext 74 3 dsRNA Reoviruses Botavirus 4 SSRNA strand Rhinovirus Yellow Fever virus Poliovirus 5 ssRNA strand Influenza Measles virus Ebola virus 6 ssRNA strand Retroviruses HIV1 Mouse mammary empey DNA intermediate tumour virus Murine leukemia virus 7 dsDNA hepatitis B virus employ ssRNA intermediate ds double stranded 33 single stranded positive polarity function as mRNA negative polarity complementary to mRNA 1 David Baltimore classification system Transcription unit 1 3 RN A AGCU nontemplate strand 5 AG CT coding strand 3 DNA TCGA template strand promoter terminator 1 upstream downstream promoter A region of DNA where RNA polymerase binds to initiate transcription terminator A region of DNA that causes RNA polymerase to terminate transcription Very rst nucleotide transcribed 1 mRNA coding strand is identical to mRNA except for Ts for Us Convention for naming the DNA strands of coding regions liilinntem plate 51 H in g i c1353 1 or ii 1 1ir 51mm iii in in i ii I i ii i it t in 1 it t any we 1 i1 rj an air insIii in 7 Template DNA 5t 3 midi 3 i i Him it it in Iii It Emit in ii 1 11 n ma n Enlist Em 1121 1 mm I1 1 it 1 w i r lii Hit it ll t It i i 5 5 quot39 input ii r 3F ITIHHA E Iniiiiii i ti I litiill militaii sfii its l i Milittlii ii i mil 1 tint Iii ii iii ij gigm miii in ii at i The nontemplate DNA strand is called the coding strand because it has the same sequence as the RNA T s instead of Us with the same 5 gt3 polarity The sequence of the template strand called the noncoding strand is complementary to the RNA and runs in the antiparallel 5 gt3 direction The Genetic Code c I The code is u C Degenerate All amino acnds except gt9 UAA Stop UGA Stop A two are encoded by more than one oodon UAG Stop UGG Trp G Unambiguous One codon never CU cc CAUHis CG U 39 39 CUC CCC CAC CGC C codes for more than one amino acnd c m WC W9 CUA CCA CAA Gm CGA A 39 nearly universal With feyv cucD ccc CAG CGGI G exceptions all codons specnfy the same amino acids in all organisms Auu Acu AAUA AGUS u sn er 1 quot we lie me me AGC c C n rv tiv Th fir ttw b A WW 0 se a e e s o ases AUA ACA AAA AGA A are usually identical when multiple AUGM V t n ACG AAGLVS AGGArg G codons specify the same amino acid GUU ecux GAU GGU u Asp GUC GCC GAC GGC C G GUA gtval GCA gtA39a GAA GGA gtGly A AUG start codon 639 cue GCG GAG GGG G UAG UAA UGA 2012 Pearson Education Inc Nearly exceptions mitochondria etc The Genetic Code Second position U C A G uuu Ph ucu UAU T UGU c e r 3 There IS one start codon AUG uuc ucc gt8 MC V uec V which Signlfles the start of the U UUA L UCA er UAA Stop UGA Stop proteinencoding sequence In mRNA UUG 6 UCGJ UAG Stop UGG Trp There are three stop codons UGA cum ccu CAU H CGU IS UAA and UAG In the genetic code c GUC gtL ccc W W cec eu gtA that sngnal the end of the proteln GUA CCA to CM GI GGA r9 n COd39ng sequence cue cce CAG CGG 8 II 395 AUU ACU AAU AGU h Asn Ser AUC Ile ACC AAC AGC A gtThr AUA ACA AAA AGA Lys Arg AUG Metstart ACG AAG AGG GUU ecu GAU GGU Asp GUC GCC GAC GGC G gtVal gtAla gtGly GUA GCA GAA GI GGA U GUG ace GAG GGG it 2012 Pearson Education Inc 0 gt O c Third position 0gtOC OgtOC Genetic code 3letter and 1letter representations gtSer u UUA L UCA UAA Stop UGA Stop uue eu uced UAG Stop UGG Trp cuu ccux CAUHis ceu u cuc ccc CAC ccc c C Leu gtPro Arg CUA CCA CAA Gln CGA A a cue ccc GAG CGG G AUU ACU AAU AGU u Asn Ser 7 Auc lle Acc AAc AGC c A gtThr AUA ACA AAA AGA A Lys Arg AUG Metstart ACG AAG AGG G GUU ecu GAU GGU u Asp GUC GCC GAC GGC c G Val gtAla Gly GUA GCA GAA Glu GGA A GUG ace GAG 666 G AGA UUA AGC AGG UUG AGU GCA CGA GGA CUA CCA UCA ACA GUA GCC CGC GGC AUA CUC CCC UCC ACC GUC UAA GCG CGG GAC AAC UGC GAA CAA GGG CAC AUC CUG AAA UUC CCG UCG ACG UAC GUG UAG GCU CGU GAU AAU UGU GAG CAG GGU CAU AUU CUU AAG AUG UUU CCU UCU ACU UGG UAU GUU UGA Ala Arg Asp Asn Cys Glu Gln Gly His lle Leu Lys Met Phe Pro Ser Thr Trp Tyr Val stop A R D N C E Q G H I L K M F P S T W Y V Polypeptides as amino acid polymers 1 2 Polypeptides 39f H 39f are polymers of amino acids quotBNCH O quot NCHCOO o Aprotein consists of one or more polypeptides Hp1H20 Amino acids 1 2 are linked by peptide bonds 39f V 39f H3N CH4c N CH c0039 II I OH 390 Peptide bond NSer GIyPheAlaValC Q3 CHSynthesizing polypeptide u H 39 Cl Polymer has polarity CHZOIl H H H CH2 H CH3 H CH Amino and Carboxyl 4 lc39 WI H3N C ll II tlilrl lfCl NrCI COO39 H 0 H H 10 H o H Amino N terminus Carboxy C terminus i Translating information from RNA into protein Sydney Brenner and Francrs Crick USGd viruses There are 4 RNA bases U C A G and they must specify 20 amino acids How many h a d A l aw 3 bases specify 539 Emily4 3lt 7i 3ltetIIA 394 w 2322ue a single amino T a T add 1 Base 2 Bases 3 Bases 4 Bases A 2base code could specify a A 3base code could specify a maximum maximum of 4 x 4 16 amino acids of 4 x 4 x 4 64 amino acids Since there are only4bases 1 U1 i c U A u U U quotii 1quot U C Iquot U A m a 1base code could specify 1 2 r 3 4 1 2 3 4 only4amino acids to U c cj to Al to cl Lc c u lc c c to c Al c c of E E E El 5 6 7 8 5 6 7 8 1 2 3 4 AM AcllAAllAel AAul lAAcJ lAAAl AAGI 4 lt 20 Not enough 9 1o 11 12 9 1o 11 12 leul G cl G A G G c G ul is c c G G Al etc 13 14 15 16 13 14 15 16 lt 20 Not enough 64 gt 20 More than enough 2011 Pearson Education Inc As there are 20 amino acids and only four different RNA bases a threebase code is the least that could specify enough amino acids it could code for 4 x 4 x 4 64 different amino acids A threebase code provides more than enough messages to code for all 20 amino acids A threebase code is known as a triplet code The group of three bases that specifies a particular amino acid is called a codon The code is read from the 5 to the 3 end The 5 end specifies the Nterminus of the polypeptide Coming soon Monday 32 HIVAIDS History cures and Penn Robert Doms The Children s Hospital of Philadelphia Perelman School of Medicine at the University of Pennsylvania Human Immunodeficiency Virus HIV Replication The HIV Life Cycle HIV medicines In six drug classes stop 0 HIV at different stages In the HIV life cycle 0 Binding also called Attachment HIV binds attaches itself to receptors on the surface of a C04 cell 0 Entry Inhibitors 9 Fusion The HIV envelope and the CO4 cell membrane fuse join together which allows HIV to enter the 04 cell quotquotquot c Fusion Inhibitors 3 Reverse Transcription Once inside a C04 cell HIV releases an HIV enzyme called reverse transcriptase HIV uses reverse transcriptase to convert its genetic materialHIV RNA Into HIV DNA The conversion of HIV RNA to HIV DNA is necessary so that the HIV can enter the nucleus center of a C04 cell and combine with the cell s genetic materialcell DNA 0 Nonnucleon reverse transcrlatue Inhlhltors NNR39I39B e Nudeeuda reverse tranurlpuu lnthltors NRTII Integration HIV produces an enzyme called integrose which allows HIV DNA to enter the CD4 cell nucleus Once inside the cell nucleus the HIV DNA is joined integrated with the 04 cell DNA 5 Transcription and Translation Once HIV is integrated into 04 cell DNA the virus begins to use the machinery oi the C04 cell to create tang chains of HIV proteins The protein chains are the building blocks for more HIV O trimaran Inhibitors the long chains of HIV proteins The smaller HIV proteins combine with HIV RNA to Iorm a new virus 0 Protease Inhibitors m 7 httpaidsinfonihgoveducationmaterialsfactsheets1973thehivlifecycle Required reading httpwwwhhmiorgbiointeractivehivIifecycle Required viewing httpwwwyoutubecomwatchveS1GODinO8w Eukaryotic cotranscriptional mRNA processing Eukaryotic structural gene I Exon1 lntron1 Exon2 Intron2 Exon3 Promoter Terminator Transcription Prem RNA 3 539 Cap gAllllnl39tappens as its being transcribed 8otPanscriptional process 1lions will be kept 439 let N h much smaller than premRNA 339 tail lntrons removed Mature mRNA is exported out of the nucleus Eukaryotic mRNA 5 capping 7methylguanosinelt 5 r 5 capping enzyme as this 5 is happining 539endof lt prokaryotic RNAchain m 5 cap 7methylguanosine covalently attached to 5 end Not encoded in gene sequence Needed for proper exit of mRNA from nucleus and binding to ribosome recognition signal for initiation of translation Note the unusual 5 to5 linkage between the 7methyl guanosine base and the 5 end of the mRNA Eukaryotic mRNA splicing Primary transcript pre mRNA 5 Exon Intron Exon Intron Exon 339 PollWmquot 1 30 31 1 04 1 05 1 46 lntrons excised and exons spliced together mRNA Powm tail 1 146 As RNA po f ifff 5iquota quot 3 i working and after 5 capping enzyme splicing enzymes set in and look for definable sequences between exons removes nucleotides and rebind the exons subsequently described well in text The proteincoding regions of eukaryotic genes are interrupted by noncoding regions To make a functional mRNA these noncoding regions must be removed Exons are the regions of eukaryotic genes that will be part of the final mRNA product The intervening noncoding sequences are called introns and are not in the final mRNA Eukaryotic genes are generally much larger than their corresponding mature mRNA Eukaryotic mRNA 3 end processing Cleayage 1035 nucleotides downstream from AAUAAA sequence PolyA polymerase Y PoyA tail How does RNA polymerase terminate in eukaryotes 3 polyA addition enzymes I 3 p0y A ta Looking forAAUAA and about 30 nucleotides downstrearr ponApolymerase adds 100200 A resudues 100200 adenine nucleotides added to 3 end 2012 Pearson Education Inc Important for nuclear export Increases stability and lifespan in cytosol and aids in translation initiation Not encoded in gene sequence Eukaryotic mRNA alternative splicing examples There are four basic types of alternative splicing alternative 5 splicesite selection a alternative 3 splicesite selection b cassetteexon inclusion or skipping c and intron retention d The rectangles in the centre represent Skipped exon preSmmgar each prem RNA the black lines span the regions that can be spliced out with the lines above corresponding to the mature mRNA shown on Ml m Win NA on the right That is the mRNA that is s sized wh or intron in d is skipped is shown on 39 the left an mm this sequence is included is shown on the right l l l idered an exon when included A ernative 539 spice site right and an intron when skipped left i i l d 1 n u 39n u Q quot I39 h p u 0 u 4quot n u p o f l i Alternatively spliced exons premRNAs mRNAs tWO related polypeptides that are dif in middle Alternative splicing Fibroblast growth factor receptor FGFRZ gene 810 12 14 1618 Exons1I a 34 139 6 11l1315l17l 19 I II I I I I ll Fibroblast Growth factor number of recepto on surface of cell that can bind these 4 Kb Majority of the gene doesn t code for exons Alternative Alternative splicing splicing mRNA Ligands FGF1O FGF7 t y F6 F8 Exterior 39 FGF5 I 39 irl l it Rig g a I Cytoplasm Fibrost rowth Fibrostrowth factor receptor 2 factor receptor 2 First isoform Second isoform Figure 919 Protein isoforms Different forms of a protein with slightly different amino acid sequences but with the same activity May be produced from different genes or from the same gene by alternative splicing OMIM Online Mendelian Inheritance in Man Online Mendelian Inheritance in Man An Online Catalog of Human Genes and Genetic Disorders Updated 23 February 2015 fgfr2 Search Advanced Search OMIM Clinical Synopses Gene Map Search History Need help Example Searches OMIM Search Help OMlM Tutorial Mirror sites useastomimorg europeomimorg Online Tutorial Within 30 seconds you can WWWOmimOrg know things about any gene on human genome connections between genes and diseases Anatomy of an eukaryotic mRNA transcript Nontemplate strand Mb r RNApolymerasebindingIsite Template strand I r r I I I I 1 Transcription 5 splice site I 3quotIsplice site Primary RNA l I o transcript I 5 5939 539 39 3 splice site n R Splicing I I I I I I I I I I I Ribosontebinding site I mARINA 5 UTR ORF 339 UTR 5 gntranslated region leader 1 Translation 3 gn anslated region trailer H2N I Polypeptide I COOH ORF Open Reading Frame The Encyclopedia of DNA Elements ENCODE Goal To delineate all functional elements encoded in the human genome ENCODE Project 32 institutions 442 people 1649 experiments 15 TB data ENCODE functional element A discrete genome segment that encodes Adefined product eg a protein or noncoding RNA or Displays a reproducible biochemical signature eg protein binding or a specific chromatin structure Nature 489 5774 7582 8390 91 1 00 101 1 08 109113 25 more in other journals The Encyclopedia of DNA Elements ENCODE l Splicing J Genome size 31 x 109 bp Gene number 20687 Protein coding genes 294 of genome Alternatively spliced transcripts 63gene Nature 489 5774 7582 8390 91 100 101 108 109113 l 25 more in otherjournals Prokaryotio translation linear polysomes Polyribosome numbers indicate order in which ribosomes attached to mRNA 5 end of mRNA Ribosome translates mRNA as it is being synthesized by RNA polymerase 391 Protein Ribosome RNA polymerase End of gene gt Start of gene 1 2011 Pearson Education Inc Multiple ribosomes attached to an mRNA form a polyribosome Eukaryotie translation circular polysomes messenger RNA o o 339 mRNA What 1s maklng these elreular 5 Cap and polyA tail covered by proteins Makes RNA more stable 39 Reinitiation of translation I p eodon dissociation akes initiationfriendly more ef cient polyAbinding protein polypeptide chain I A B Translation Initiation elongation and termination A g Gene quot 5 mRNA 3 Ribosome Gene 5 3 mRNA AUGCUCGGAUACCGUCAU GUGUGCAGGCAUUCAUAA LJ Start Codon Stop Codons UAA UAG UGA Translational initiation Prokaryotes ShineDalgarno base pairing ribosome binding site RBS 30 ShineDalgarno Start sequence codon Eukaryotes 5 capdependent scanning Small subunit binds to cap and then scans until it finds Kozak sequence AUG is start codon WGCC CCAUGG Methylated cap Initiation site 1 Small subunit binds to methylated cap Mecca CCAUGG 2 Small subunit migrates to initiation site 4 W60 Kozak sequence Response of E coli to the appearance of lactose in the absence of glucose 4 Most proteins involved with lactose use are degraded E coli DNA 1 Lactose becomes available in the environment of the bacterium lactose acts as an inducer a molecule that stimulates the expression of a specific gene 3 The bacterium readily uses the lactose until it is gone Lactose Bgalactosidase Breaks down v 4 quot v lactose 4 q a 4 Lactose permease Transports lactose into A v the cell 2 Due to gene regulation the bacterium produces more of the proteins lactose permease and Bgalactosidase Lactose permease and galactosidase Bgalactosidase side reaction Allolactose OH H OH H 4 Bgalactosidase Lactose permease H OH H OH Galactose Glucose F E coli Prokaryotic gene regulation Lactose operon Regulatory gene lac operon f N f N Promoter IacZ gene I lacY gene I lacArgene Operator 1 5 3 5 3 laclmRNA IaomRNA l l l l39 q 39 7 i 39 l l Galactosrde Trans 39 233 B galacms39dasei ipermease acetylase 1 2012 Pearson Education Inc m A bacterial operon is a cluster of genes under the transcriptional control of one promoter The polycistronic mRNA encodes sequences for 2 or more genes Each gene in the operon has its own ribosome binding site Allows regulation of a group of genes with a common function lacZ lacYacA are encoded in the operon lacl is transcribed from a separate constitutive always on promoter m lacl encodes a protein called lac repressor that represses transcription of the Lao operon DEED Prokaryotic gene regulation Lactose operon RNA polymerase blocked from transcribing lac operon Repressor bound to operator DNA racz gene lacY gene IacA gene IacImRNA l a Lactose absent repressor bound to operator operon repressed In the absence of lactose the repressor remains bound to the v operator and RNA polymerase Is therefore prevented from moving Acme f rm down the lac operon and transcribing its genes ot repressor In the absence of lactose lac repressor protein binds to a DNA sequence the lac operator lacO This prevents the vast majority of transcription of the polycistronic mRNA An example of negative regulation There is a small amount of transcription which ensures that there will be some LacZ and LacY protein present in the cell Prokaryotic gene regulation Lactose operon RNA polymerase bound to promoter W gt DNA aggro lacZ gene 5 3 5 IacImRNA l IaomRNA l Inactive form RNA polymerase transcribing the operon lacY gene I lacA anew l l Galactoside Trans permease acetylase of repressor b Lactose present repressor not bound to operator operon derepressed In the presence of lactose the repressor is converted to its inactive form which does not bind to the operator FINA polymerase can therefore move past the operator and transcribe the lacZ IacY and lacA genes into a single mRNA 2012 Pearson Education Inc In the presence of lactose Lactose is transported into the cell by LacY permease protein Some lactose is converted to allolactose by LacZ The allolactose binds lac repressor protein preventing the repressor from binding to the operator DNA This derepresses transcription of the operon cis acting elements and transacting factors DNA or RNA sequence elements that affect the expression of adjacent typically downstream genes are said to act in cis Latin for on the same side cisacting element lacO DNA RNA polymerase blocked from transcribing lac operon racz gene lacY gene IacA gene transacting factor lac repressor protein In contrast diffusible factors often proteins or RNAs that regulate other genes are said to act in trans Latin for on the opposite side Lac operon genetics Table 23l Genetic Analysis of Mutations Affecting the lac Operon Phenotype with Inducer Absent Phenotype with Inducer Present Line Number Genotype of Bacterium B galactosidase Permease Bgalactosidase Permease 1 IPOZY 2 IPOZY 3 IPOZY 4 1 P O Z Y 5 IP39OZY 6 ISPOZY 7 I POZY P Pm The defective permease exhibits su icient biological activity to transport minimal amounts of lactose into the cell thereby permitting induction of the lac Operon it 2012 Pearson Education Inc Note The text uses 239 and Y39 to denote mutations that produce low but nonzero levels of Bgalactosidase and permease Why is this important for understanding the mutations 4 0 constitutive operator 6 IS super repressor 5 p39 loss of function promoter 7 I39 loss of function repressor Score Number of Students 0 3 gt O 00 L O L 3 L 4 20 25 3o 35 4o 45 5o 55 60 65 7o 75 8O 85 90 95 100 AVG 805 i 171 BIOL 121 33915 Exam1 Translation Initiation elongation and termination Ribosome Gene 5 339 mRNA AUGCUCGGAUACCGUCAU dif types at 5 and 3 ends GUGUGCAGGCAUUCAUAA Start Codon Not all genes are expressed all the time regulated often at transcription level Stop 00d0n5 2 of genome codes for polypeptides U AA A lot is switches turning genes on and off Translation hardly ever starts at beginning of mRNA Untranslated region both holld regulatory elements A Operarate differently in eukaryotes and prokaryotes Only 1 that starts at beginning of RNA Prokaryotic Ribosomal binding site Eukaryotic 5 cap bind and scan Translational initiation in Pm kartDies ad39tiz39t astz t 308 ribosomal subunit F2 Ribosomebinding site ShineDalgarno sequence ribosome binding site R88 539 1 ShineDa39gamo Start Initiator tRNA mRNA AUG sequence codon Start codon e Initiator tRNA and mRNA bind to the Small subunit binds to 8D sequeno Figure 91 3 Introduction to Genetic Analysis Tenth Edition I in large ribosome through GTPSI Formymethionine tRNA of start 39r lm 721 W 39 mRNA binding site 303 initiation complex 0 508 ribosomal subunit K becomes bound to the GTP hydrolysis 30 initiation complex Formyl group NIH ii CH3 S CH2 CHz II C O H NFormymethionine 70 initiation complex Reading frame Start codon defines the reading frame 5 AUAAGGAGGUUACGAUGCAGCAGGGCUUUACC 3 Met Gln Gln Gly Phe Thr I u c V Thiking about all mRNA but e WU ucu Phe prokaryotes U WC quot0 UUA UCA UAA Stop UGA Stop A Must translate approp protein UUGLeu m m StopUGG Tquot G Use complementarity to adjs seduence cu ccu u More Info than Just translated c we ccc 39 c CUA Lequot CCA gtPr A a cue coca CAG cars 6 AUU ACU AAUAsn AGUSer u Auc Ile Acc AAC AGC c 39739 A gtThr AUA ACA AAA L s AGA Ar A AUG Metstart ACG AAG y AGG 9 G GUU ch GAUA GGU u GUC GCC GAC Sp sec c G Val gtAla Gly GUA GCA GAA GI GGA A GUG ace GAG GGG G 1 sequence and 3 reading frames DNA 5 CAT CAT CAT CAT CAT CAT CAT CAT RNA 5 AU CAU CAU CAU CAU CAU CAU CAU frame1 his his his his his his his his 5 C AUC AUC AUC AUC AUC AUC AUC AU frame2 4 ile ile ile ile ile ile ile 5 CA UCA UCA UCA UCA UCA UCA UCA U frame3 ser ser ser ser ser ser SE Potential to translate into 3 frames More potential information Usually one reading frame is used and initiated with AUG Some viruses have small chromosomes use overlapping reading frames to generate 2 dif polypeptides Translation reading frames reading direction for sequence of top DNA strand 3 N ile leu phe arg val ile arg pro thr arg asn phe thr arg C read39ng 2 N tyr phe ile ser ser asn ser thr leu asn ala lys leu his leu thr C 1 N leu phe tyr phe glu phe asp Ieu lys arg glu thr ser leu asn C frames DNA 1 C lys ile glu leu leu glu val lys phe ala phe ser lys val N read39ng 2 C ile lys asn arg thr ile arg gly val arg phe lys val arg N frames 3 C asn lys ser thr asn ser arg leu arg ser val glu ser leu ser N 4 reading direction for sequence of bottom DNA strand Coding strand When sequencing don t know Which is coding strand either could be Each frame is interrupted With stop and go Figure 8 52a Molecular Biology of the Cell Garland Science 2008 Try this Go here Random DNA Sequence Generator 1 Generate 60bp sequence Enter values and click button Size of DNA in bp 60 GC content between 0 and 1 050 Generate Sequence CGTAGTGAGACTTACATGTTCGTTGGGTTCACCCGACTCGGACCTGAGTCGACCAAGGAC l Assign 5 and 3ends Make double stranded convert both strands to RNA Translate both strands in all three reading frames Assign 5 and 3 end See if you can find an open reading frame 1 httpwwwfacultyucredummadurorandomhtm Translation Open Reading Frames ORFs How to annotate DNA Best guess of What region codes for reading direction for sequence of top DNA strand gt 31 llllllllj l 1 1 11 111111 l reading l frames 2 1 l 111 1 L1 I l l 1 IlI I S 3 DNA 3r 539 1ll I mil I I I 2l Hill 1 T 1 11 Dirt TIIIITH T II 1 ii i 3 Ill 1 lt reading direction for sequence of bottom DNA strand I l 500 base pairs reading frames vertical blue lines stop codons gray boxes ORFs red boxes ORFs that begin with AUG codons Figure 8 52b Molecular Biology of the Cell Garland Science 2008 Types of mutations Single basepair substitutions are called point mutations Mutations can also result from insertions or deletions of multiple bases and chromosome rearrangements Basepair substitutions can create a Missense mutation DNA G A A G T A C T T i C A T mRNA G A A gt G A Protein Glu gt pc Nonsense mutation DNA T T A T A A A A T A T T mRNA u u A gt u A Protein Leu gt Stop Silent mutation DNA C C C C C A G G G G G T mRNA c c c gt c 05539 Protein Pro gt Pro Basepair insertions or deletions can create a Frameshift mutation insertion ATGCAAGTTTGAC TACGTTCAAACTG DNA ATGAAGTTTGAC TACTTCAAACTG mRNAAUGAAGUUUGAC gtAUGAAGUUUGAC Protein Met Lys Phe Asp gt Met Stop VJ Missense Nonsense Mistakes in DNA polymerization or insults to the genome ay to predict what happens to the gtlypeptides when these happen lllllll Illllll chromosomes Insertion D3333 lt i l l I I l l Deletlon Duplication EIllll ICED Em Inversion L Translocation reciprocalr I I I I I I I DNA of nonhomologous lllllll k a Mutations affecting one base pair G 2012 Pearson Education Inc b Mutations affecting long DNA segments initiation termination Prokaryotic transcription Transcription unit 5 DNA 39I A 3 3 H 5 J Promoter Un ound DNA Terminator 0 Binding of RNA polymerase and local DNA unwinding 3f 5 6 Initiation of RNA synthesis 3 Sammie specific for one type of RNA NTps cell or another Template Strand 0 Elclaset arwb tt terms 5 Initiation highlighted by particular 339 e promoter 5 3 Termination signal RNA palymeraseOl 0 Termination of RNA synthesis 5 3 3 5 539 339 RNA transcript 2012 Pearson Education Inc Prokaryotic transcription initiation Polymerase movement Unwinding of DNA f 77777 gt f i r 1 I 391 Ilr II 1 I 1 It Jr I 5 j 5 r 51 b39 1 1 I i i t F RNA POLYMERASE Coding of DNA Nucleotide being added to the 3 end of the RNA RNA 39 I 3 1 quot RNADNA hybrid region is 2012 Pearson Education Inc 5 to 3 Where does transcription start Where does it start Recognizes particular sequences look at a IDiomotOi sequenQNhich DNA strand serves as the template Promoter sequence defines two key decisions Promoter DNA Coding 35 sequence 10 sequence Startpoint strand r 1 5quot Lquot wquot r v 5I Template strand Transcription 5 gt339 RNA 2012 Pearson Education Inc Can t land on either orientation Can tell which direction it has to go Prokaryotic promoter a E coli promoter alignment Gene 539 UTR 6 0 AUG Transcrlptlon 539VWWVgt Promoter Coding sequence of gene b Strong E coli promoters tyr tRNA rm D1 rm X 1 rm DXE2 rm E1 rm A 1 rm A2 Consensus sequences for most E coli promoters Figure 87 1 ATG J TCTCAACGTAACAC TGCGGCG o 39 39lll v ix GCGCCCC CTTCCCGATAAGGG GATCAAAAAAATACTGCAAAAAA o o TTGGGATCCCGCGCCTCC TTGAGACGACAACG ATGCATTTTTCCGCTCT CCTGAo oGCCGACTCCCGCGCCTCC TCGACACGGCGGAT CCTGAAATTCAGGGTCTGAAA o o GAGGAAAGCGACo GCCAC TCGCGACAGTGAGC CTGCAATTTTTCTAVCGGCCTGCG 5 E 39ii39fililT TCGACACGGCGGAT TTTTAAATTTCCTCTGGCCGG illl r li GCGCCACC CTGACACGGAACAA GCAAAAATAAATGCTCTGTAG o o CGGGAAGGCG TGCACACC CGCGCCGCTGAGAA 1 1517bp I TTGACAT TATAAT I 35 10 Pribnow box In troduction to Genetic Analysis Tenth Edition 2012 W H Freeman and Company Certain sequences are the same consensus sequence a generalized base sequence derived from closely related sequences where each position in the consensus sequence is found in the majority of sequences at that position E coli RNA polymerase promoter binding it I 5 jgtoiltyjfEEXQ V Biochemical evience LI Transcription Promoter region start site I 35 1o 539 AGTTAGTGTTGATAGAAGCACTCTACCTCAATAGGTCCACGG 339 II A 17 3909 I I Mild effects II39 on transcription Onebase IIIII deletion Ambase II I change Severe effects I I I on transcription usually affects usually affects RNAPbMdm RNAPbMdm also can affect Genetic evidence template melting E coli RNA polymerase binds to one side of the doublehelix i i i i mimimimimiquot Polymerase noW nt insert into 611202 major grooves to start Number of nucleotides in between matters insertion l 2bp l l i mutation 3 quotmimmimmmr Genomes Chromosomes DNA 23 pairs of chromosomes Fluorescence light micrograph Karyotype The entire chromosome complement of an individual organism or cell as seen during mitotic metaphase 121815 Scanning electron micrograph Largest chromosome 1 Smallest 21 50 million base pairs DNA has territories in nucleus certain chromosomes near one another likely contribute to the expression of one another 3 billion base pairs 322 Giga bases n m Genome Reference Consortium 1 849118 assembly GRCh38p2 12814 2 3 198295559 4 190214555 5 181538259 8 170805979 7 159345973 8 145138838 9 138394717 10 133797422 11 135088822 12 133275309 13 114384328 14 107043718 15 101991189 18 90338345 17 83257441 3 18 80373285 10 um 19 58817818 Human mitotic chromosomes 20 64444167 FISH karyotype 21 48709983 47 Mb 316 Gb haploid genome 22 50398 839468 Alberts et al 2008 Figure 410 X 15610401895 Home 11111111711117 n bi n m n h Y 5712271415 U m Chromosome compaction and 3D nuclear arrangement This 11mm in purely diagrammatic The two ribbons symbol the chum and the hod voulul rods the pain If hue holdln the chains together 9 vertical line math the bre axis two plutonium sugar CU Tertiary structure I W 30nm chromatin J Secondary structure 1 I I Nucleus with distinct territories 7 vb39rbl gr I 10mm chromatin Primary Structure 316 Gb haploid genome 3 feet of DNA in each cell httpwwwncbinmnihgovprojectsgenomeassemnygrchumandata Genome architecture analysis reveals chromosome territories j quotA I arranging P39fu t t T39r d a JS EQUp a S massage quot own 39 6 Deeper RNA scqucncmgr jut 39 v 0 121815 Kalhor et al 2012 Nature Biotechnology 30 90 98 2012 Chromosomal theory of inheritance Mendel s observations Today Particulate factors Genes Two members of a gene pair segregate Alleles Experiments in Plant Gene pairs assort independently Meiosis Hybridization 1865 Are there physical objects in a cell that correspond to Mendel s hypothetical factors Chromosomes What is the chemical nature of chromosomes DNA and protein Crossed plants Chromosomal theory of inheritance Natun Reviews I Genetics 2 Theodor Boveri links chromosomes and heredity by discovering chromosome reduction in meiosis 1889 3 Others showed that chromosomes contain DNA and protein 1 Walther Flemming describes chromosomes and deduced the sequence of chromosome movement during mitosis 1882 Nature Reviews Molecular Cell Biology 2 72 75 2001 Nature Reviews Genetics 9 231 238 2008 Griffith s bacterial transformation experiments Late 1920s Frederick Griffith was working with Streptococcus pneumoniae Strains that secrete capsules look smooth and can cause fatal infections in mice S Strains that do not secrete capsules look fro ugh and infections are not fatal in mice R O Asmmdeggibraworg Pritt 1918 Pandemic between 5 anc 10 died Spanish flu H1 N1 The most devastating flu pandemic in recent history killing more than 500000 people in the United States and 20 million to 50 million people worldwide Many from secondary illnesses pneumonia Strain R Griffith s bacterial transformation experiments Treatment Conclusion Smooth strains 8 with capsule are Control Type 8 cells fatal Injected living 39 are virulent type s bacteria Capsule prevents immune into mouse system from killing bacteria Living bacteria found in blood Control T R H Injected living 328 Rough strains R Without capsule are type R bacteria nOt fatal into mouse NO lVlng bacteria found In bIOOd Conclusion A substance in the Sstrain transforms the Rstrain to the lethal Iconttrczjlzh t Heatkilled niec e ea t e 8 cells killed type s an benign If mice are injected With heatkilled bacteria into type 8 they survive mouse Injected living quot Living type MiXing live R With heatkilled 8 kills the tYpe R and t i quot v 7 K R cells mouse healkilled have been Blood contains living S type type S bacteria transformed bacteria into mouse into virulent type 8 cells Transformation Conclusion A substance in the S strain K Sgi39rfmtype S transforms the Rstrain to the lethal phenotype Transformation In general Any alteration in the properties of a cell that is stably inherited Genetic transformation classical A process by which a donor DNA molecule is taken up from the external environment and incorporated into a recipient cell germ line transformation Bio has its own lingo Microinjection of DNA into embryo and subsequent incorporation into DNA of germ line cells thereby yielding transgenic organisms malignant transformation Genetic changes in animal cells associated with cancers viral transformation Malignant transformation of animal cells induced by viral genes Avery MacLeod and McCarty 1944 biochemical experiments Hypothesis A purified macromolecule from typeS bacteria which functions as the genetic material will be able to transform typeR bacteria into typeS First experiment Types cells Ki then disrupt amp fractionate Polysaccharides Lipids RNA Protein DNA 0 TypeR cells Avery et al second experiment Experimental level Conceptual level 1 Purify DNA from a type 8 strain This i involves breaking open cells and separating the DNA away from other components by centrifugation DNase i RNase Protease Type R cells 2 Mix the DNA extract with type R bacteria Allow time for the DNA to be taken up by the type R cells converting a few of them to type 8 Also carry out the same steps but add the enzymes DNase RNase or protease to the DNA extract which digest DNA RNA and proteins respectively As a control don t add any DNA extract to some type R cells Add an body DNA DNase RNase Protease Control 3 Add an antibody a protein made by the immune system of mammals that aggregates type R cells that haven t been transformed Remove gt TypeR 4 Remove typeRceIIs by x cells centrifugation Plate the remaining bacteria if any that are in the supernatant onto petri plates Incubate overnight Type 8 cells in supernatant Type R cells in pellet Iquot i Centrifuge Avery et al second experiment D 2 Mix the DNA extract with type R q bacteria Allow time for the DNA to be taken up by the type R cells converting a few of them to type 8 Also carry out the same steps but 39 add the enzymes DNase RNase or protease to the DNA extract which digest DNA RNA and proteins Add respectively As a control don t add A B C antibody any DNA extract to some type R cells contm39 DNA DNA DNA DNA DNase RNase Protease z I 1 I I I I z I I I I I I z II I I A I I 1 l Plate cells from supernatant I If I I z I z I I I I I 1 I 1 I I z I I I 5 THE DATA A quot r V A r gt quot If C p lt r quot 1 DNA extract 3 DNA extraa RNase contro39 DNA extract DNase DNA extract protease no bacteria bacteria no bacteria bacteria bacteria Genetic material ToDo list Contain the information necessary to construct an blueprint entire organism Pass from parent to offspring and from cell to cell during cell division her39tab39e Be accurately copied Stable Account for the known variation within and mutable between species Watson and Crick s insights during model building Insight Data DNA is helical Rosaland Franklin Bases are internal Helices have opposite polarity AT 11 and oc 11 a Erwin Chargaff Ph0t051 Purine pyrimidine complementarity WC modeling a interestingly DNA from different cells of one species exhibits the same base composition Base composition varies among species Base composition 30 content Streptomyces coelicolor 72 Human 42 Bacteriophage T4 35 Drosophila 44 EcoH5196 httpwwwhhmiorgbiointeractivegreatdiscoveries sciencedoubIehelix Required viewing httpwwwyoutubecomwatchv1vm3odUm Fgampfeatureyoutu be 91012261500 MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid wish to suggest a structure for the salt of deoxyribose nucleic acid DNA This structure has novel featums which are of considerable biological interest quotIt has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic materialquot a Doublestranded DNA Antiparallel strands run parallel to each other but with opposite polarities Nucleic acids have a sugarphosphate backbone The sugarphosphate backbone of RNA 539 end of 5 The sugarphosphate backbone of a o nucleic acid nucleic acid is directional one end has an unlinked 539 carbon and the other end has an unlinked 339 carbon The nucleotide sequence is written in the 539 gt 339 direction This reflects the sequence in which nucleotides are added to a growing molecule This nucleotide sequence comprises the nucleic acids primary structure joined by phosphodiester anage What would change in the figure 3 end of nucleic acid if it were a new nucleotides are added I to the unlinked 339 carbon 3 1 representatlon Of DNA 2011 Pearson Education Inc Nucleic acids are built with nucleotide monomers Nucleic acids are polymers RNA and DNA Monomer is called a nucleotide Made up of phosphate group a fivecarbon sugar either ribose or deoxyribose and a single or double ring of carbon and nitrogen atoms known as a base a Nucleotide Phosphate group is bonded to 5 carbon of sugar Nitrogenous base 139lt group 3 2 Nitrogenous 5carbon base is bonded to sugar 139 carbon of sugar b Sugars 1 5 1 i i0quot H 3 02 H OH OH Ribose in RNA Deoxyribose in DNA 2011 Pearson Education Inc c Nitrogenous bases Cytosine C Uracil U in RNA Thymine T in DNA J Y Pyrimidines NH2 N Purines are larger than pyrimidines Guanine G Adenine A Purines Helix ththanded lefthanded Doublestranded DNA helix JGHN TEMPLETGN FUN TII N EUP P39ETIHG EE39IEHEEIHEE39EETIHG IN THE BIG 39IILUEETIHWE Genetics httpwwwtempletonorgwhatwefundcorefundingareasgenetics accessed 2515 The maximum yield of aerobic respiration is 38 ATPS per glucose for some prokaryotes and eukaryotes 39 The maximum ATP yield for oxidative phosphorylation from one glucose ONAD 10H ZFADH so2 34ADP 3er1 gt10NAD 2FAD 12H20 34ATP gt 10 X 10 Hl translocated gt 30 ATP 2 X 6 Hl translocated gt 4 ATP Energy yield for aerobic respiration Glucose 686 kcalmol ATP 10 kcalmol cell environ 10 X 38 380 kcalmol for ATPs 380 686 55 0o recovered Emrgyyied r rmem a m Gmore 68 6 6511 7770 ATP 70 Aral77ml 2 X 70 20 m moforATPr 20 686 3 recomrea Including the reactions of glycolysis and the TCA cycle give s 38ADP38 38ATP 602 6co2 6H20 Emrgy farawayfrom car e gz39 e 250o recomrea The actual yield of aerobic respiration is less than 38 ATPs per glucose GLYCOLYSIS PYRUVATE PROCESSING Cytosol Mir Oxidative Total 100 AND cnmc ACID CYCLE matrix pho s yleld P Gl col 81 2 ATP 2 ATP y y 2 NADH gt 6 ATP 6 ATP 0 NADHZXZ 6x ATP Pyruvate NADHquot 39 39gt 3 ATP 6 ATP GILquot X X2 to acetyl 1 CoA Krebs ATP 2ATP NADH X2 5x ATP cycle 3 NADH gt 9 ATP 18 ATP X 2 FADHZ gt 2 ATP 4 ATP In reality the value is lower PMF used for other things membranes leak complexes 1 and 111 can transfer a few electrons to 02 sometimes takes energy to transport electrons from cytosolic NADH produced by glycolysis into the matrix etc Change in free energy AG in kcalmol m I I I L I I I O 01 00 N C O o O O O 39 O O O O O O 1 1 1 1 1 Oxidative phosphorylation Oxaloacetate 6 2011 Pearson ducauon Inc membranes leak protons anabolic processes reducing power involved in producing molecules many cases In the absence of oxygen anaerobic conditions the TCA cycle and oxidative phosphorylation would both stop for humans and many other organisms colysis NAD CoA a Ace 39 CoA w 3 V 39 v r quot v cu vv 39CQQQ tut 6v c39c V V v v quot Vt 1 0 f g w rn vn A n n n n A n n 39 970799990 1 enuneeeae inhan ow iaym u COA 37 O C o o a dsqg r q I q aw y I I in 777 Oxaloacetate Citrat 39 quot 1 9 V a 39 39 39 M 711quot M i if lutm 7 lquot l l if F4 945 Biow 7279 ed Campbell and Ram 2005 PM I n 5quot m 9 39 l 39r 30 FAD 09 NAD Anaeroblc condltlons no 02 summate No terminal electron acceptor 0 co2 No way to pass electrons from NADHFADH2 No way to generate proton gradient No way to do oxidatiye phosphorylation No FADNADregeneration Stops TCA cycle Starts fermentation But many prokaryotes can use alternatlve electron acceptors for respiration Less ef cient than 2 but more ef cient than fermentation N03 N02 trimethylmine oxide gt ecoli 4 391 Under normal conditions the electron transport chain is coupled to ATP synthesis why Uncouplers can abolish this coupling important in brown fat cells used to generate heat more when baby bears have a lot all energy Rm NADH and FADH2 not ccinverted to ATP so goes to at gt hibernation 39 llows continue e transport an O2 consumption without ATP 2i synthesis the system energetically would stop the more H on one side the harder it is to pump more across 39 u o o w 39 w f y 39 N ip mrmpvw 39 With an uncoupler I 39 V Qquoti39 39 quot R 1 q ocooooooovW vzzzzizi i 1 41 It l l fl p39 3quot r i um i n k H move down their gradient Lowers the proton gradient H don t pass through ATP synthase No oxidatiye phosphorylation g t ls li 39i o i 00quot y r i r f 39V 17 I tquot Fzg 975 Biology 7277 ed Campbell and Reece 2005 Panama Uncouplers do not have to be channels one example is dinitrophenol introduced in the 1930 s as a weight loss drug people died from it gt Other drugs chemicals can affect the process in other ways Cyanide inhibits complex IV The insecticide rotenone inhibits complex I Photosynthesis Photosynthesis synthesis from light Plants take in C02 and release water and 2 Light is required Al lbl t5 IMEKIINwrbo n Photosynthesis for plants Light 6C02 6H20 a C6H1206 602 Don t forget plants and many other photosynthetic organisms also catabolize sugars Plants have mitochondria Many organisms can convert solar energy into Chemical energy plants do this in chloroplasts Plant cell Outer quot 39 ENERGY TnAusnuc ou nEAcTIous Chloroplast membrane Light gm Inner 13 u h Thy39akmd w Electron membrane 9 transport and harvestl Proton lntermembrane Punian space Thylakoid membrane quot Thylakoidlumen H H4 CYTOSOL ATP synthesis H H H CARBON ASSIMILATION REACTIONS 4 Pm th phosphates Sucrose Starch name39s 1 synthesis ucrosol snatch 777 Beteeer LVord 0f 96 Cell 8277 ed Hardin 62 52 2072 Pearson Atoms can absorb photons of speci c wavelengths The energy from the photon boosts an electron into a higher energy state Exc ed state Absorption Photon of photon by 39H molecule 0 Ground Wavelength nm 1 itquottitquotT77551t f3939fiih fi state E Visible Ii B Photon Electron Nucleus Ground State Excited state UFE THE SCIENCE OF BIOLOGY Eighth Edition Molecules that absorb speci c wavelengths usually in the visible range of the spectrum are called pigments Intensity of the sun s radiation at the Earth s surface Absorption l 300 400 500 600 700 800 900 1000 Wavelength nm Chlorophyll agreen Phycoerythrin red Chlorophyll bgreen Phycocyanin blue Bcarotene and lutein orange Key 2 Pearson Education Inc absorb the different wavelengths available in environment Light gathering molecules are organized into photosystems and lightharvesting complexes Photosystems are in the thylakoid membrane in plants They contain pigments that absorb light antenna pigments and pass the energy to chlorophyll molecules in reaction centers Where electron transport occurs Light harvesting complexes LHC are accessory complexes that also absorb light and pass the energy to Photon the photosystems thereby increasing the ef ciency of light collection The LHC are mobile in the membrane and can move to take advantage of changing light Resonance conditions transfer Antenna pigments Photosystem Thylakoid membrane lt Electron acceptor Reaction center chlorophyll Electron transfer 777 Beaeeer LVord 0f 96 Cell 8277 ed Ham39m 62 52 2072 P65275072 Electrons can follow one of two paths the Zscheme or cyclic photophosphorylation why so 16 Zscheme of photophosphorylatio ATP and NADPH 12 0398 h c 9 g 0 X o o4 Q 3 Wm E 2 NADPquot 2H s e V o 8 cu a C 2 E Photon 04 m 6 27333 Spht H20 08 2 12 Release 02 Protons released into thylakoid lumen Photosystom ll Photosystom I 50 V reducing power 04 08 12 778 Bet 673 LVord 0f 96 Cell 8277 ed Hardin 62 52 2072 P66175071 Cyclic photophosphorylation allows for a balance of NADPH and ATP synthesis Electron excitation Photon Protons pumped into thylakoid lumen No water is oxidized not 02 released No NADPH is generated 7770 Becker World of Zae Cell 8277 ed Ham39m 62 52 2072 Pearson The ETC of photosynthesis generates NADPH and ATP suns 4 Photons 4 Photons 2 STROMA 2 NADP 3 4i 0 In ow Photosystem I 4 ATP synthase complex 4 Cytochrome be complex Photosvstem TlIYLAKOID LUMEN 779 Beckeer LVord 0f Zae Cell 8277 ed Hardin 62 62 2072 P65273072 Photophosphorylation is analogous to oxidative phosphorylation Mitochondrion Chloroplast MITOCHONDRION CHLOROPLAST STRUCTURE STRUCTURE Intermembrane space Thylakoid space or Ele ton 3qu 5 Inner membrane 0ft M 1 1 Thylakoid membrane 202 I DIDIIIOIOIO39 Matrix Key I Higher H H Lower H 7076 Biology 7277 eel Campbell and Reece 2005 P66175071 The Calvin cycle enables conversion of carbon from C02 into more reduced compounds such as sugars and carbohydrates that can be metabolized by plants animals and other organisms carbon xation O Synthesis of From the atmosphere 3phos glyceratel macromolecules O 0 0 O O O O O I Ribulose15 bisphosphate From the light reactions Occurs in the chloroplast stoma Glyceraldehyde3 Fixed carbon for synthesis phosphate of other organic molecules 777 7 Becker World of 96 Cell 8277 ed Hardin 62 52 2072 P65275072 The Calvin cycle For one turn 3C02 9ATP 6NADPH5HZO gt glyceraldehyde 3 phosphate 9ADP 6NADP8Pi Carbon xation The 6 Carbon intermediate following C02 addition has never been detected and is probably very unstable Ribulose15 blsphosphate a H C OH H C OH H lz o H THE CALVIN CYCLE CC1 Ribulose bisphosphate carboxylaseloxygenase rubisco CC2 Phosphoglyceroklnase CC3 Glyceraldehyde3phosphate dehydrogenase CC4 PhosphoribulokinasePRK rubisco is the most abundant protein on the planet 3 RibuloseSphosphate A series of reactions to regenerate rlbuloseSphosphate 7772 Ber 673 LVord of 96 Cell 8 ed Ham m 62 52 2072 PedVJO Should look familiar H C OH H c o H Glycerate 13blsphosphate 6 Glyceraldehydes phosphate 0quot One out of every six molecules Five out of every six molecules Biosynthesis of sucrose starch or other organic molecules Photorespiration competes with photosynthesis when the enzyme rubisco uses 2 instead of C02 Appears to be an unavoidable side effect Reaction with carbon dioxide during photosynthesis gt Favored when RuBP cc2 RUblsco two 3phosphoglycerate 39 C02 concentration is high 39 02 concentration is low 1 CO2 and 02 act as competitive inhibitors for each other used in Calvin cycle Favored when 39 02 concentration is high Reaction with oxygen during photorespiration C02 concentration is low RuBP 02 m one 3phosphoglycerate one 2phosphoglycolate l 1 used in Calvin cycle when processed C02 is released and ATP is used gt Inef cient 25 becomes C02 the remaining 75 recycled into glycerate Undoes photosynthesis C4 and CAM plants minimize photorespiration by separating carbon xation from rubisco CO2 incorporated into three carbon organic acids carbon xation PEP carboyxlm 39 CO2 Organic cid 4C Mesophyll atmosphere CAM spatial separation temporal separation ame CO2 incorporated into fourcarbon I l r a Night organic acids 1 4 jomata open carbon xation Day 39 Stomata close Organic acids c release CO2 to Calvin cycle Suar At high temp C02 less soluble than 02 Also stomata close in dry conditions PEP carboxylase has a lower Km for C02 than RuBisCO 02 is a very poor substrate for this enzyme 7020 Biology 7 ed Campbell cmd Reece 2005 P66273072
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