Cell Bio Final Exam Study Guide
Cell Bio Final Exam Study Guide BIOL 30603
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This 95 page Study Guide was uploaded by Mallory Notetaker on Thursday April 28, 2016. The Study Guide belongs to BIOL 30603 at Texas Christian University taught by Dr. Akkaraju, Dr. Misamore, Dr. Chumley, Dr. McGillvray in Spring 2016. Since its upload, it has received 144 views. For similar materials see Molecular, Cellular, and Developmental Biology in Biology at Texas Christian University.
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SS cell bio ﬁnal exam study guide Professor sent out this study guide -questions on earliest material will come from this Molecules of life- Chemistry Properties of different types of intermolecular interactions- covalent, non-covalent, hydrophobic/ hydrophilic. Understand the chemical basis of these interactions. Covalent: -strongest bond -takes lots of energy to break, permanent -bond formed by sharing electrons Non-covalent: -ionic bond- dispersal of magnetic attractions -ex: proteins and their substrates -weak -hydrogen bonding - important in holding proteins together and forming shape -electrostatic interactions: 2 charged atoms come together (a salt) -water dissolves things that have electrostatic interactions Hydrophobic/hydrophilic -two drops of oil in water, they want to come together to reduce the amount of surface area that is touching the water -no charge but come together Properties of different macromolecules in the cell: Sugars, proteins, lipids, steroids, nucleic acids. How does structure influence the behavior of these molecules? Sugars- linked by glycosidic bonds (created by condensation) -3 carbon sugar = triose, 5 carbon sugar = pentose, 6 carbon sugar = hexose -glucose = hexose aldose -Sugars can form ring structures, they switch back and forth from their straight chain to ring -enzymes will look for certain orientations of functional groups on their substrates (sugars) -because of the different functional groups on the carbons, glucose is different than galactose -mannose is a sugar that used to tag proteins in the cell, called glycosylation -the order in which the sugars are added to the proteins, tells where to send the protein and this is speciﬁc because of their distinct properties Proteins -made up of amino acids linked by L-peptide bonds (covalent) -carboxylic acid groups linking -amino acids are made up of an amino group, carboxylic acid, R group, and a hydrogen attached to a carbon -the R group or side chain can be basic, acidic, uncharged polar, or nonpolar -the R groups will eventually determine the nature of the amino acids which will in turn determine how the protein is folded. Lipids -Long carbon chains, carboxyl group at one end and hydrocarbon tails off them -Hydrophobic because they are non polar -fatty acids have energy in them so the cell breaks these to make ATP -double bonds in chain = unsaturated -no double bonds = saturated -triglyceride: 3 fatty acids stuck on glycerol by ester linkages -phospholipids -3 carbon backbone and add a phosphate -one side is polar(phosphate) and the other is non polar (fatty acids) -one fatty acid is saturated and one is unsaturated (important) Steroids -long chain fatty acids folded back in on itself -storage of energy -need cholesterol in cell membranes to keep ﬂuidity Nucleic Acid -made of nucleotides Pyrimidine- base in a hexagon shape Thymine Cytosine Uracil Purine - base in a almost naphthalene shape Adenine Guanine -they are phosphorylated and can attach up to 3 phosphates on them -phosphates add on the 5’ of the sugar -phosphates are connected by phosphodiester bonds -Base adds on the 1’ of the sugar -2 types of sugars (pentoses) are used in nucleotides -Beta-D-ribose -in RNA -Beta-D-2-deoxyribose -in DNA Bioenergetics/ Enzymology How does the Second Law of thermodynamics influence biological systems? -says that the disorder in the universe can only increase -SO reaction that increase order, require energy What are Free Energy and ΔG, and how do they determine what kind of reactions can occur in a system? Free energy: a thermodynamic quantity equivalent to the capacity of a system to do work. ΔG: What is a coupled reaction? -one reaction is unfavorable so it is coupled to a favorable reaction -reaction 1 has a + delta G so it is coupled with reaction 2 that has a negative delta G -the breakdown ofATP is coupled with the production of sucrose so that it will occur What are some of the molecules biological systems use to move reactions in an unfavorable direction? Enzymes -lower activation energy of reaction -the reaction rate levels off after adding more and more substrate because all the enzymes are already in use What are energy carriers? -high energy intermediate where energy is trapped in the phosphate -ATP - carries phosphate -NADH, NADPH, FADH2 - carries electrons and hydrogens -Acetyl CoA- carries acetyl group -Carboxylated Biotin - carries carboxyl group What is Km? How does it change with the affinity of a reaction? Km: an afﬁnity constant -the higher the afﬁnity is, the lower the Km is -Understand Vmax is the max amount of substrate -use intersection of 1/2Vmax and Km to ﬁnd the rate of reaction -be able to use 1/2Vmax to derive Km point and then compare that to the afﬁnity of another graph What kind of chemical events occur during ‘condensation’and ‘hydrolysis’in metabolic reactions? Through condensation, the body joins two molecules together by releasing water -energetically unfavorable Hydrolysis is the opposite, breaks down molecules -energetically favorable -The water that is lost in joining molecules together, comes from parts of phosphates and the inorganic material itself (OH from phosphate) Shape and structure of proteins Levels of protein structure- primary, secondary, tertiary, and quarternary. primary: made up by amino acids bonded by peptide bonds secondary: occurs when the sequence of amino acids are linked by hydrogen bonds, alpha helix and beta sheet tertiary: overall 3D shape of an entire protein molecule, bend in a way to reach max stability quaternary: made up of protein subunits, form larger aggregate protein complex What are the bonds that hold monosaccharides, amino acids, fatty acids and nucleotides together? monosaccharides- glycosidic bonds amino acids - peptide bonds fatty acids - held to glycerol by ester linkages phosphates - phosphodiester bonds nucleotides - phosphodiester bonds through condensation What is feedback inhibition? it avoids the buildup of wasteful or toxic metabolites -when you have a build up, the products bind to the enzyme at an allosteric site (somewhere other than the active site) and reduce the enzymes activity -conformational change -in some cases, a Cofactor (allosteric inhibitor) can cause a conformational change that increases the afﬁnity of the enzyme for the substrate, being an activator What kind of modifications are used to control protein expression? Regulation of protein function Protein Kinases: add phosphates to enzymes to activate them -but sometimes phosphorylating an enzyme can inhibit it -phosphorylation does not automatically mean activating -the added phosphate group will cause the protein to adapt in its folding and will thereby change the function of the protein Phosphotases: remove phosphates from enzymes Genomes/ DNA packaging and replication What is the evidence derived from genome sequencing and comparison that suggests a common origin for plants and animals? E.g., What is the evidence that shows a common ancestry between gorillas, chimpanzees and humans? These species are not related, they split a long time ago, but the genes are still really similar -chimps are the closest to humans -theres very few differences between human and chimp gene -and when there is differences, they are conservative differences meaning the changes in the nucleotide sequence doesn’t change the protein that is coded for -order of phylogenetic splitting: orangutan, then gorilla, then chimp and humans -this is based on the percent difference in nucleotide substitution How do you read an evolutionary tree such as the one shown in this figure? What is the structure of DNA? the double helix -sugar phosphate backbone -nucleotides attach in the middle adenine with thymine guanine with cytosine How is DNApackaged into chromosomes? DNAwraps around histones to create nucleosomes (beads on a string) and then chromatin is formed from packed nucleosomes (30nm) and then condenses more and more What is a nucleosome? Do NOT memorize the individual histones? -DNAwrapped around a histone How does histone modification control gene expression? Modifications of histones- leads to changes in gene expression -can be inherited -but after a couple of years, in your germ cells, the modiﬁcations reset -all the little histones have tails and thats where the modiﬁcations happen - need to know that the modiﬁcations that happen to histones are methylation, acetylation, and phosphorylation, don’t happen at the same time -modiﬁcations of histones acts as a code to tell the nucleosome what to do What is a SNP? Single Nucleotide Polymorphism can cause changes in humans, to look different -small changes within single nucleotide level -if they happen within the regulatory sequence or protein coding regions can cause a change within the function of the protein, a reason why we have differences What are the enzymes involved in DNAreplication? What is the function of topoisomerases? Origins: initiator proteins bind here and break hydrogen bonds between DNAstrands RNAPrimer: a short length of RNAproviding a base paired 3’end, starting point for DNA polymerase DNApolymerase: makes the complementary DNAstrand in the 3’to 5’direction, uses parent DNAtemplate DNAprimase: makes the RNAprimer DNAhelicase: unwind DNA DNAligase: seals breaks in the phosphate-sugar backbone, joins okazaki fragments Topoisomerases -release the super coiling tension created by DNAreplication topoisomerase I: breaks the DNAand attaches it to the tyrosine in its active site, allows to uncoil, then reattaches it topoisomerase II: clips one of the strands and pulls it over the tangled one, takes the energy when it breaks it and uses it to reattach it DNA to protein What is transcription? -production of RNAfrom DNA Know: coding strand, template strand coding strand: the strand thats sequence corresponds with the codons that are translated into proteins, aka it has the same base sequence as the mRNAproduced Template strand: sequence that is being copied to make mRNA Know: RNApolymerase I, II and III and its function, general transcription factors, activator proteins, enhancers. RNApolymerase I: transcribes rRNAgenes RNApolymerase II: all protein coding genes, miRNAgenes -needs TATAbox RNApolymerase III: transcribes DNAstrands that have a different starting sequence other than the TATAbox What are the three steps of RNAprocessing? Capping- 7-methylguanosine added to the 5’end of the growing RNA Polyadenylation- addingAAAAAAto the end of the strand poly-Atail: stabilize RNA, keep from getting degraded Splicing RNAsplicing only occurs in eukaryotes -there are exon mixed with introns -splicing: taking out the introns mediated by snRNPs snRNPs look for intron-exon junctions; help in cleaving of those sites -10% of mutations can effect splicing Know the enzymes involved in protein synthesis: tRNAsynthetases, Ribozymes, Ribosomes, start codons. tRNA -one end is called anti codon and binds to the mRNA -and other end has the amino acid Amino acyl tRNA synthetase: job is to attach one amino acid to the tRNA -these are super speciﬁc -this is important because the tRNA could go off and attach that wrong amino acid -so it proofreads by checking the sequence of the tRNA and the amino acid -has two active sites, synthesizing and editing -checks twice and removes wrong amino acid if need be Ribozymes- rRNA (RNA that has enzymatic activity) Ribosomes build protein -ribosomal RNA has enzyme activity, so it is called a ribozyme (RNA enzyme) -E site, P side, A side -goes in the 5’ to 3’ direction -ribosomes are stuck on the ER -In the active site ribosomal RNA presents an Adenine to help with building of the protein -eIF2: binds to the small ribosomal complex and forms the pre initiation couples -that goes and binds to the cap (eIF-4E) -starts to slide down the mRNA looking for the start codon -when it ﬁnds it the large ribosomal subunit binds and begins making proteins -energy for protein synthesis comes from GTP -Initiation, elongation, termination Start codon: AUG which codes for Met Know the function of the proteasome. How are proteins targeted for degradation? How does a cell know that a protein needs to be degraded, it tags it with polyubiquitin -then it is dragged over to the proteasome, like a hollow barrel and inside there are proteases that chew up the protein -ubitiquin tag is not degraded, and recycled -this is important in the cell cycle because not only bad proteins are degraded but proteins that are not needed at the time Control of gene expression Why control gene expression? Examples of gene regulation -All cells contain all the genes in the genome -different cells in the body and turn on different genes -taking a skin cells and turning into the embryonic cell -in diseases you want to determine which genes are on and off in a cell What is an operon? How can it be regulated? Operon: group of genes that turn on and off a gene; an efficient way to make the genes that are required to make one protein Can be controlled through positive or negative control Positive regulation -when the regulatory protein is present, the operon is on (activator) Negative regulation -when the regulatory protein is present, the operon is off (repressor) Lac Operon Lactose: operon on, removes repressor, need to speciﬁcally not have glucose What is RNAi? RNAInterference post-transcriptional regulation of gene expression -a biological process in which RNAmolecules inhibit gene expression, typically by causing the destruction of specific mRNAmolecules -microRNAis short RNAthat folds on itself to make a double stranded structure -and they are complementary to similar RNA -formation of RISC -knock down DNA expression by preventing RNA expression by degrading the mRNA Complete Akkaraju material (only questions above will be tested on ﬁnal) virus is non-living outside a cell, needs a cell to live -viruses are compact packages of DNA or RNA encased in protein Ancestral cell is 3.5-3.8 billion years ago -prokaryotes most closely resemble ancestral cell (no nucleus) the cell is the functional unit of all free-living organisms Prokaryotes can use inorganic materials (sulfer, CO2) to generate energy instead of only depending on oxygen. Prokaryotes are the most diverse group of organisms on the planet (more variety of species) -eukaryotes have a symbiotic relationship with prokaryotes to help us survive What is the consistency of the cytosol of the cell? -gel-like, snot -cytosol is the liquid part of the cytoplasm (area between nucleus and cellular membrane) -If things have to move they have to be transported across the membrane -organelles are ﬁxed in space Nucleus has DNA, and it is distributed unevenly (nonuniform), some packed tightly some packed loosely -euchromatin: loosely packed -heterochromatin: tightly packed, permanently silenced -after replication the DNA condenses (so it is not chromatin) in the metaphase Endoplasmic Reticulum- helps in the transportation of proteins and in the processing of proteins, proteins get modiﬁed here Golgi Apparatus- protein motiﬁcation and protein transport Plasma membrane- holds the cell together Plant- cell wall (helps resist osmotic change) Organelles Microscopy is how we discovered them (ﬁrst step in cell biology) light microscope: ﬁrst type used, uses light, cannot see organelles source of radiation: light through lenses detection technique: natural pigment or stains absorb light differently resolution: 1000x magniﬁcation up to .2um Fluorescence: tag different parts of cell, can see things inside the cell source of radiation: methods of illumination and electronic image processing, two ﬁlters for the light to pass through detection technique: different wavelengths illuminate ﬂuorescent dyed parts of cell resolution: 20 nanometers Confocal: look at speciﬁc organelles, creates 3-D image source of radiation: laser beam detection technique: pinhole aperture in the detector allows only ﬂuorescence emitted form this same point to be included in the image. resolution: can see organelles Transmission Electron: cell cut into thin sections, uses beam of electrons as equivalent for light detection technique: stain specimen with electron-dense heavy metals that locally absorb or scatter the electrons resolution: 1nm Scanning Electron: scatters electrons off the surface of cells to observe the surface in great detail, 3D image detection technique: resolution: 3nm-20nm What are the different types of organelles? Mitochondria function: generators of chemical energy for the cell, produce ATP, cell respiration nature of their membranes: they have two membranes, came from bacteria that were engulfed by a eukaryotic cell (created a symbiotic relationship between eukaryote and bacteria) Chloroplasts function: photosynthesis membranes: two membranes, also have internal stacks of membranes containing chlorophyll Endoplasmic Reticulum function: make materials that get exported, synthesis, modify, and transport proteins membranes: contain ribosomes (rough ER) that convert RNA into proteins, continuous with the membranes of the nucleus Golgi Apparatus function: modiﬁes and packages molecules made in ER membranes: stacks of ﬂattened membrane enclosed sacs Lysosomes function: break down food Peroxisomes function: breaks down fatty acids, etc and turns them into hydrogen peroxide then further breaks down that toxin The cytoskeleton: made up of… -microﬁlaments: made up of actin and myosin, thin threads that spread across cell to give it shape -Microtubules: larger than microﬁlaments, hollow tubes, help transport things across cell, important during cell division (pulls apart chromosomes during metaphase) and the separation of DNA Model Organisms: these things we discover in these organisms we can apply them in humans Saccharomyces cerevisiae: Yeast: has the same genes that work in eukaryotes Arabidopsis thaliana- helps study plants Drosophila melanogaster- ﬂies Caenorhabditis elegans: sea elegans: helps unravel the development of eukaryotes -nematode, helped us understand apoptosis (a form of programmed cell death by which surplus cells are disposed of in all animals) -important in cancer research Mus Musculus- zebra mussels, good for studying vertebrates Questions: protein-protein interactions are not covalent bonds covalent bonds are permanent and it takes energy to break it the resolving power of a microscope is limited by the wavelength of radiation used -the ability to see detail in the cell Electron microscope can see DNA and ribosome Organelle has both an outer and an inner membrane -mitochondrion -endosymbiosis: one little membrane organelle got swallowed by another Mitochondria -has own genome -able to duplicate -divide on a different time line from the rest of the cell -it cannot live outside the cell and the cell cannot live without the mitochondria so mitochondria and the cell are endosymbionts Bonds Strength: Covalent- takes energy to break, strong Noncovalent: ionic hydrogen bonds vanderwaals -Electrostatic bonds help proteins bind Water structure -cohesive nature of water gives its unusual properties -high surface tension -high speciﬁc heat -high heat of vaporization -it dissolves other polar molecules because it is polar -hydrophilic will dissolve in water, hydrophobic is opposite -ionic and polar molecules are hydrophilic, they dissolve -the hydrophobic (nonpolar) parts of proteins stay on the inside and are sheltered from the aqueous environment of the cell Sugars -Aldose: sugar with an aldehyde on it -Ketose: contains ketone group -know that a three carbon sugar is a triose, and know pentoses and hexoses -Glucose is a hexose aldose -know the monosaccharides chart really well, even the speciﬁc names glyceraldehyde ribose glucose dihydroxyacetone ribulose fructose Sugars can form ring structures, they switch back and forth from their straight chain to ring -know the numbering of the carbons on the ring formation slide -enzymes will look for certain orientations of functional groups on their substrates (sugars) -example: glucose is different from galactose -mannose is a sugar that used to tag proteins in the cell, called glycosylation -the order in which the sugars are added to the proteins, tells where to send the protein and this is speciﬁc because of their distinct properties -glycosidic bond is when sugars are linked together -glycosidic bond created by condensation, taking out water -as soon as they are linked, the beta and alpha form of the hydroxyl group is frozen -3 linked together, trisaccharide -chitin are sugars linked together and is very hard to break down -you can add other groups in place of hydroxyl groups on sugars -know and be able to name the substituted sugars glucuronic acid lucosamine N-acetylglucosamine glucose + glucose = maltose glucose + galactose = lactose glucose + fructose = sucrose -Oligosaccharides: a small number of repeating units -Polysaccharides: lots of repeating units -can be branched Lipids Long carbon chains, carboxyl group at one end and hydrocarbon tails off them Hydrophobic because non polar Fatty acid bonds have energy in them, the cell break them and use them to form ATP -fatty acids can also be bad for the cell so they are stored in the peroxisome and broken down there Very long chain fatty acids = VLCFA Double bond in the chain creates a kink= unsaturated unsaturated = good saturated (butter) = bad for you - because of the kinks of unsaturated fatty acids, they are more ﬂuid and don’t stick to your arteries as well as fatty acids do -triglyceride: 3 fatty acids stuck on glycerol by ester linkages -when you go into the clinic they look at your levels of triglycerides (can be bad) Membranes are made up by lipids -phospholipids and glycolipids form self-sealing bilayers -Naturally occurring fatty acids interacted with water and formed the ﬁrst bilayer trapped inside of it DNA making ﬁrst life Phospholipids -3 carbon backbone and add a phosphate -one side is polar(phosphate) and the other is non polar (fatty acids) -one fatty acid is saturated and one is unsaturated (important) -The unsaturated fatty acids in the membrane make it more ﬂuid and less rigid Ignore isoprene slide Steroids -long chain fatty acids folded back in on itself -storage of energy -need cholesterol in cell membranes to keep ﬂuidity -testosterone- male sex hormone Amino Acids -Stucture: 4 things attached to an alpha C -amino group -carboxyl group -hydrogen -side chain (R) (20 side chains) -they are grouped according to whether their side chains are -basic -acidic -uncharged polar -nonpolar Basic: sucks up hydrogen (amine group), reducing acidity acidic: gives hydrogen into the system -anytime you see a charge on a molecule, it is polar Peptide Bonds -can form peptide bonds by linking the carboxyl group to the amino group of another -all peptide bonds in our body are the L form not D -Protein contain exclusively L-amino acids -peptide bonds are formed by condensation and are ﬂexible because single bonds Nucleotides Pyrimidine- base in a hexagon shape Thymine Cytosine Uracil Purine - base in a almost naphthalene shape Adenine Guanine -they are phosphorylated and can attach up to 3 phosphates on them -phosphates add on the 5’ of the sugar -phosphates are connected by phosphodiester bonds -Base adds on the 1’ of the sugar -2 types of sugars (pentoses) are used in nucleotides -Beta-D-ribose -in RNA -Beta-D-2-deoxyribose -in DNA The nature of the amino acids determines how the protein will fold phosphates are bonded by phosphodiester bonds shorter chains of the nucleotides are oligotides base is attached to carbon number one and phosphate is on carbon number 5 a covalent bond between two atoms is formed as a result of the sharing of electrons Cells need ribose to build nucleotides Weak non covalent chemical bonds -two proteins come together and have a high afﬁnity to each other -things in the cell are constantly moving and can randomly ﬁnd a substrate and act on it Vander walls attractions -at short distances, any two atoms show a weak bonding interaction due to their ﬂuctuating electrical charges Hydrogen Bonding: -when an H is between two electron-rich atoms (oxygen) -strongest when the 3 atoms are in a line - is important in holding proteins together and forming shape electrostatic attractions: between two charged atoms -water breaks up electrostatic interactions, dissolving the compound If two things have a high afﬁnity for each other the rate of dissociation will be low hydrophobic interactions -two drops of oil in water, they want to come together to reduce the amount of surface area that is touching the water -no charge but come together Reactions in the cell are broken down into smaller steps because it is easier to move something uphill one step at a time Catabolic pathway: break down; release energy through oxidative pathways anabolic pathways: build up The second law of thermodynamics: universal tendency of things to become disordered -The disorder in the universe can only increase -increasing overall entropy of the universe Reactions that tend to increase order will require energy, the universe naturally slides to non order energetically unfavorable reactions can occur only if it is coupled to a second energetically favorable reaction -negative delta G coming from the synthesis of ATP is coupled with the positive delta G of the synthesis of sugar on test will give a reaction and give the free energy of the components and you need to come to a conclusion about whether it will happen G products - G reactants = delta G Misfolded proteins can have devastating consequences to the cell and molecules -alsheimers example Enzyme activity -After adding more and more substrate, it levels off because all the enzymes are already in use -Km: an afﬁnity constant -the higher the afﬁnity is, the lower the Km is -Understand what Vmax is the amount of substrate -use intersection of 1/2Vmax and Km to ﬁnd the rate of reaction -be able to use 1/2Vmax to derive Km point and then compare that to the afﬁnity of another graph -Competitive inhibitor will bind to the active site and prevent enzyme from acting on it’s normal substrate -Noncompetitive inhibitor binds to somewhere other than the active site and change the enzyme so that it can still not bind to its normal substrate -be able to derive Km from V vs S graph -substrate + inhibitor line has a higher km so it has lowered the enzymes afﬁnity for its substrate Coupled reactions using energy carriers -the breakdown of ATP can cause your body’s temp to go up -the breakdown of ATP is coupled with the production of sucrose so that it will occur glucose + fructose = sucrose In energy transfers, a high-energy intermediate is formed where the energy is trapped in the phosphate and then it can be used to help an unfavorable reaction occur (picture said condensation step) NADP+ is an energy carrier than can be reduced to NADPH NAD is also an energy carrier, know that the structure is made up of two nucleotides and a proton is added on the nicotinamide Acetyl CoA is the carrier used in fatty acid synthesis, energy found in the bond between acetyl and CoA -energy lies between the bond of the acetyl group and the Coenzyme A **know the functional group associated with each carrier and know the examples the book gives for each** ATP Carries: phosphate Use: exchange of chemical energy, intracellular signaling pathways NADH, NADPH, FADH2, Carries: electrons and hydrogens Use: in making cholesterol Acetyl CoA Carries: Acetyl group Use: fatty acid synthesis Carboxylated Biotin Carries: carboxyl group Use: transfers carboxyl group to a pyruvate to make oxaloacetate (citric acid cycle) S-adenosylmethionine Carries: methyl group Uridine Diphosphate Glucose Carries: glucose Biosynthesis Through condensation, the body joins two molecules together by releasing water -energetically unfavorable Hydrolysis is the opposite, breaks down molecules -energetically favorable -The water that is lost in joining molecules together, comes from parts of phophates, the inorganic material itself (OH from phosphate) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Shape and Structure of Proteins lecture Secondary Alpha helix or beta sheet shape is determined by the bond angles Tertiary -lots of different forces make the protein fold into the most stable or lowest free energy state (hydrogen bonds give this structure some stability ) -fold with non polar parts on the inside Chaperone proteins: assist in folding -lots of different shapes for a protein to be but it needs to be in the correct shape to do the right function -Many chaperone proteins are called heat shock proteins -if you shock a cell by exposing it to a higher than normal temperature it will start to make these proteins to try to protect the cell from the heat shock, the proteins in the cell will denature so chaperone proteins assist in folding them back -Example: one that looks like a box, you put polypeptide in the box, cap it, the amino acids on the sides of the box will start to pull and unfold the polypeptide, until the polypeptide gets in the right position, then when it is in the correct form the box can’t pull it apart anymore because it is in its most energetically favorable form and its packed super tight -Important: GroEL and GroES are the chaperone proteins that re-fold misfolded proteins -Many degenerative neurological diseases are caused by proteins that fold incorrectly and cause the death of the neuron Proteins can have regions that are unstructured so the protein can have some variability in form Coiled-Coil -A coil that binds to another protein and coils up -Two helices will bind in a way that protects the hydrophobic region -Spontanious Two proteins come together: -can get a dimer, just 2 proteins together -Helix -Ring -there are non covalent bonds that occur in proteins together -one type of covalent bonds is disulﬁde bridge -Cysteine’s come close together and allow for disulﬁde bond to form -NOT SPONTANEOUS -increases protein stability -only way to get the bond to break is by reducing it and heat ex of reducing agent: B-mercaptoethanol Spontaneous protein conﬁguration -virus capsule -because of the high afﬁnity of these 3 proteins for each other means the assembly doesn’t need an input of energy -sometimes need help of scaffold protein Binding Sites -the amino acids line the active site -the amino acids make hydrogen bonds with the substrate (cyclic AMP) -this is the reason it is very speciﬁc and binds very closely The point mutations in proteins can change the binding sites and reduce the afﬁnity of the enzyme for its substrate -cancer -cancer mutates a lot so inhibitors become unable to regulate the enzymatic activity Cyclic AMP activate proteins -can induce a conformational change Lysozyme function: cleaves the glycosidic bond in sugars -found in tears and saliva -antibacterial protein -binds to sugar on the surface of bacteria and cleaves the glycosidic bond -need glutamine amino acid and aspartic amino acid and water for this to happen -enzyme force the substrate into a transition state (bond strain) which makes it more able to be cleaved -Need Asp 52 and Glu 35 and water to accomplish this Proteins -can force two substrates together and make a reaction occur between them -an bring charges together which create partial negative/positive charges that favor reactions Feed back Inhibition avoids the buildup of wasteful or toxic metabolites -when you have a build up, the products bind to the enzyme at an allosteric site (somewhere other than the active site) and reduce the enzymes activity -conformational change -in some cases, a Cofactor (allosteric inhibitor) can cause a conformational change that increases the afﬁnity of the enzyme for the substrate, being an activator example: Cylic AMP works by forcing hydrogen bonds -from Km graph should be able to know if it is a allosteric inhibitor, etc Regulation of protein function Protein Kinases: add phosphates to enzymes to activate them -but sometimes phosphorylating an enzyme can inhibit it -phosphorylation does not automatically mean activating -the added phosphate group will cause the protein to adapt in its folding and will thereby change the function of the protein Phosphotases: remove phosphates from enzymes Beta sheets are very strong -if you make a protein with a lot of beta sheets, its harder for it to come out of that conformation, hard materials are made with a lot of beta sheets -example: spider silk Genomes -human genome = 25,000 genes -not testing on gene ancestor slide -The genes that are similar between human gene and mouse gene are mostly parallel, the order of the exons on both chromosomes are the same, these two genes originated from a common chromosome Transposons: sequences in our genome that can move around, independent sequences that can move from one chromosome to another -ﬁrst discovered in corn (maize), different kernels have different colors due to this The beta-globin gene cluster: is a retro virus because it goes from RNA to DNA 700 million years ago there was an ancestral globin gene (leghemoglobin) then it split into alpha and beta globin genes Relationships can be recognized across vast phylogenic distances -the older the gene, the more similar u will ﬁnd it to other genes These species are not related, they split a long time ago, but the genes are still really similar -chimps are the closest to humans -theres very few differences between human and chimp gene -and when there is differences, they are conservative differences meaning the changes in the nucleotide sequence doesn’t change the protein that is coded for -order of phylogenetic splitting: orangutan, then gorilla, then chimp and humans -this is based on the percent difference in nucleotide substitution If a mutation happens within the exon the cell will die or the DNA machinery would ﬁx itself, mutations happen randomly, mutations that happen in the exon part is bad, if it happens in the intron the mutations stays in the genome because it doesn’t get discarded Are there important mutations in the introns? -yes, for splicing, they tell the snRNPs to cut and where On the slide that compares species getting farther and farther away from humans, green represents the conserved changes -CTFR gene: prevents liquid from building up in ur lungs -in all of the species the exon part is the same for all of them, even all the way to fugu(blowﬁsh) -you can use these gene comparisons to identify genes that are important You accumulate mutations over time Shufﬂing exon: one exon from one gene jumps to another Transposons: serve as agents from genome evolution Horizontal transfer: bacteria can inject genes into each other leading to antibiotic resistance A point mutation in the lactase gene -forever ago humans stopped drinking milk so the gene to digest milk was only turned on early in life -ten thousand years ago we domesticated animals and drinking milk -then there was a variance in the gene and that got spread around because the people who could digest milk were healthier, could reproduce more children -this mutation occurs in the regulatory gene Exon shufﬂing slide: because functionally unrelated genes have similar components, they originated from an ancestral gene that underwent exon shufﬂing Euchromatin: DNA is currently being used in the cell, loosely packed Heterochromatin: is not being used in the cell so it is tightly packed normally a 30 nm chromatin ﬁber, but in metaphase it condenses into a 1400 nm chromosome Remember the different types of histones, H2A, H2B, H3 -the loosening of the genes is controlled by histones Information from review questions How can transposons change chromosomes? -they cause a deletion in part of chromosome -To change the stability of a protein, the mutation has to be in the amino acid sequence, not the regulatory DNA of a gene Forming of histones Nucleosomes are formed by histones -*remember names of histones -core histones H2A H2B H3 H4 -linker histone = H1 We identify different chromosomes with color, FISH (ﬂuorescent in situ hybridization) Hybridization: important way to detect sequences on DNA -use probe, it binds to the speciﬁc sequence you want it to -ﬂuor is attached to probe, wavelength will light up ﬂuor so that we can identify the sequence’s location …And from this we learned that essentially chromosomes are actually organized in the nucleus Gene expression can alter the localization of genes in the genome -genes move in the nucleus when it needs to be expressed -in the middle of the cell are transcription factories -in nuclear neighborhoods (on the sides of cell) different things happen -either expression or gene silencing -3.2 billion nucleotides in our genome -prokaryotes have a much similar gene expression -1.5% of our DNA sequence is exons, protein coding sequences -this is such a small number for how complex we are -Because bacteria/prokaryotes genomes are way smaller in number than ours, they don’t have many non coding sequences (introns) like we do Actual size of average human gene is ~27 base pairs, but we only need 1.3 base pairs to encode the amino acids 50% of the genome is repeated sequences, many of them are transposons, we don’t know their function yet -not all are transposons, they started as transposons and jumped into genome and now have no function 50% of the unique sequences and about 20% of that is protein-coding -the rest is regulatory sequences -some are microRNA 150 bp, they control gene expression, they don’t make protein they work as RNA Single nucleotide polymorphisms (SNPs) -can cause changes in humans, to look different -small changes within single nucleotide level -if they happen within the regulatory sequence or protein coding regions can cause a change within the function of the protein, a reason why we have differences Transcriptome: controls all the transcripts that come from your DNA catalome: aims to catalog all the proteins in your genome ineractome: aims to catalog all the interactions between proteins in your genome DNA replication and packaging Centromere: important for the attachment of the mitotic spindle Replication origin: where replication begins, hundreds of them Telomere: end of the chromosome, has proteins bound to it, repeats in it, it is important for the cell to know where there is a break and an actual end Histones: -regulate gene expression (important!!) -Form the nucleosomes The histones have to be persuaded to let go of the DNA and to do this, they have to be modiﬁed (nucleosome remodeling leads to the opening up of the DNA) -occurs because of the remodeling complex, uses ATP -it is a reversible the process, loosen or tighten Multiple histones that form the nucleosome -all the little histones have tails and thats where the modiﬁcations happen - need to know that the modiﬁcations that happen to histones are methylation, acetylation, and phosphorylation, don’t happen at the same time -modiﬁcations of histones acts as a code to tell the nucleosome what to do -methylation for example could cause a conformational change because of charge addition (nonpolar) -Methyl alone = heterochromatin formation, gene silencing -Methyl + Acetyl = gene expression -Phosphoryl + Acetyl = gene expression -don’t memorize for exam -The removal would require a de-acetylate Information from review questions: The Classic “ beads on a string” structure is the most decondensed chromatin structure possible and is produced experimentally. Which chromatin components are not retained when this structure is generated? linker histones Modiﬁcations of histones- leads to changes in gene expression -can be inherited -but after a couple of years, in your germ cells, the modiﬁcations reset X inactivation -one of the X chromosomes in females needs to be bundled away and never used -and all the daughter cells will have the exact same modiﬁcation -prevents over expression of genes on the chromosomes DNA replication DNA polymerase proofreads its work RNA polymerase does not proofread -A and T -C and G DNA polymerase -has two active sites -Polymerizing -Editing -It detects the mistakes by feeling the diameter Knotty Problems -Because DNA is a double helix, it generates a coiling tension—-> supercoil -When you pull the two strands apart, you are relaxing the coiling tension at one end but increasing it at the other -the DNA polymerase will eventually have to stop if you don’t release the tension -To solve this problem, you have to cut the DNA and the cell relaxes the super coiling -Enzymes that do this are called topoisomerase I: breaks the DNA and attaches it to the tyrosine in its active site, allows to uncoil, then reattaches it Topoisomerase II: clips one of the strands and pulls it over the tangled one, takes the energy when it breaks it and uses it to reattach it Inhibitors -if you inhibit topoisomerase, the cell will die -Fluroroquinolones - used to kill bacteria topoisomerase -Doxorubicin: lymphoma, sarcoma (kills cells) -Genistein After DNA synthesis, you have to go back and replace primers with DNA -every round of cell division the chromosome is getting shorter and shorter because the ﬁrst strand does not have another OH group to easily close the gap between the fragments so the cell chews up the gap Replicative cell Senescence: cells getting old -We don’t want this to happen to your germ cells -to prevent this, there is a cellular clock -Telomerase adds a bunch of repeats on the end -uses RNA as a template to extend the gap and then puts down primer and adds complementary DNA -Also this occurs in cancer cells, they turn on the promoter of the telomerase -Somatic cells turn off telomerase, as a way of controlling cell division -Radiation kills your germ cells Dyskeratosis congenita -defects that prevent telomerase from being turned on Random questions from class Mutations in how many genes directly involved in telomerase extension could cause diseases such as DC? -not just caused by one gene Mutations -DNA is constantly damaged -So, it needs to be constantly monitored and repaired -As you get older, theres an exponential relationship between age and getting cancer -more and more mistakes will accumulate in your DNA -The diameter of DNA changes when there is a mutation or an abnormal base Types of mutations Depurination: break the sugar between the base Deamination: converts the amine group to a ketone, which means cytosine —> uracil UV radiation: DNA absorbs this energy and can create random bonds like a bond between Thymine bases next to each other, creating a Thymine Dimer -this causes a bulge in the backbone, changing diameter of helix -also can happen if one of the bases is a cytosine How to ﬁx mutations -Base excisions repair: go in and clip the mutation with polymerase and then seals it with ligase, remove one nucleotide (abnormal bases) -Nucleotide excision repair: if you have a dimer, you remove a whole stretch of nucleotides and puts better DNA down Xeroderma pigmentosum patient -happens when you can’t repair the DNA -Defective DNA repair enzymes Which strand to repair? -Abnormal bases: something other than ATGC is there and it is an easy ﬁx -Abnormal base pairing: if U pops up, it will remove it and put a C -Probability of wrong base in a mismatch example: G:C-Me —> G:T (the T has a higher chance of being removed) -Nicks in daughter strand, the enzyme goes looking for a nick in the strand to tell which one is the new strand so it will replace the base on that strand Question: Hyperactive telomerase can cause… cells to become resistant to replicative cell senescence What is the difference between exon shufﬂing and alternative splicing? -alternative splicing only occurs on RNA -have one gene making on strand of RNA and you take speciﬁc exons from that strand Transcription: production of RNA from DNA -not all RNA is turned into proteins Messenger RNA: code for proteins Ribosomal RNA: formt he core of the ribosome’s structure and catalyze protein structure RNA polymerase is not speciﬁc for any part of your DNA -its afﬁnity to bind to DNA is very low -needs a helper called sigma factor (increases the afﬁnity) -sigma factor binds to the promoter (TATATTT box) -always reads the template strand 3’ to 5’ -reaches the terminator sequence -these sequences can’t be found in eukaryotes just in bacteria -The promotor (TATA box) are not found on the RNA, only on the DNA -promotor starts -10 and the RNA strand is made starting at the +1 place on the template strand -In eukaryotes, translation happens outside the nucleus and transcription happens inside -bacteria everything happens in the same place, so it can happen at the same time Genes can be transcribed in different directions in the genome -but always goes from 3’ to 5’ RNA polymerase I: transcribes rRNA genes RNA polymerase II: all protein coding goes,miRNA genes, Transcription by RNA polymerase II -TATA box (promotor) is the minimal amount of sequence needed, meaning if you mutate the TATA box, transcription will either stop or not be able to produce a useful amount of RNA -general transcription factors for DNA polyermase II will be called TFII -TFIID: will bind to the TATA box and then other transcription factors start assembling around it -There is a tail on the RNA polymerase II called CTD and it is phosphorylated, makes it sticky to other proteins -Every DNA strand that has a TATA box will be transcribed by DNA polymerase II, if the starting sequence is different, it will be transcribed by DNA polyermase III -The starting step (initiation) is the rate determining, everything after is very fast -a gene can be read by multiple RNA polymerase II to produce a lot of RNA DNA binding proteins make contact with the nucleotide sequence -amino acids on proteins interact with the bases -also makes the DNA bend -mutation in the sequences would cause the afﬁnity of this interaction to become weak RNA processing -only happens in eukaryotes -happens in nucleus -Transcription in eukaryotes is tightly linked to RNA processing -go back and get notes The RNA factory -changing the phosphorylation sequence on the tail of the RNA polyermase II, the RNA processing proteins jump from the tail to the RNA sequence -in this order: -capping factors -splicing factors -polyadenylation factors Order of things on a strand of RNA: Cap open reading frame poly-A tail: stabilize RNA, keep from no interruptions getting degraded Capping: structure 7-methylguanosine added to the 5’ end RNA splicing only occurs in eukaryotes -there are exon mixed with introns -splicing: taking out the introns mediated by snRNPs snRNPs look for intron-exon junctions; help in cleaving of those sites -10% of mutations can effect splicing Review Questions -as you are exposed to things in life, your epigenetic change, the modiﬁcations on your histones diverge -RNA polymerase doesn’t need a start codon, a start codon is only used in translation not transcription -operons can be regulated by repressor proteins -opertaor is not a protein its a piece of DNA, it binds to the tryptophan repressor and shuts off the operon snRNPs are a mix of protein and RNA, it hybridizes with the intron exon junctions -U1 and U2 bind to the junctions and they are brought together by UA and U6 -multiple snRNPs interact with each other to splice -splicing always goes from 3’ to 5’, you can skip exons -from one genes you can get ﬁve different RNA strands and get ﬁve different proteins from one gene Splicing mutation -causes microcephalic osteodysplactic primordial dwarﬁsm -a mutation in a gene encoding snRNA Poly-adenylation -here it moves from pre-mRNA —-> mRNA -moves out of the nucleus, and the mRNA nucleopore checking system that makes sure only mature mRNA comes out -when it comes out of the cell, initiator factors bind to the mRNA -in PROKARYOTES, polycistronic mRNA, they have multiple start codons so one mRNA can make 3 proteins Eukaryotes -separation of location for transcription and translation Prokaryotes -not separate Translation: mRNA —-> protein -genetic code is decoded by tRNA -one end is called anti codon and binds to the mRNA -and other end has the amino acid Amino acyl tRNA synthetase: job is to attach one amino acid to the tRNA -these are super speciﬁc -this is important because the tRNA could go off and attach that wrong amino acid -so it proofreads by checking the sequence of the tRNA and the amino acid -has two active sites, synthesizing and editing -checks twice and removes wrong amino acid if need be Ribosomes build protein -ribosomal RNA has enzyme activity, so it is called a ribozyme (RNA enzyme) -E site, P side, A side -goes in the 5’ to 3’ direction -ribosomes are stuck on the ER -In the active site ribosomal RNA presents an Adenine to help with building of the protein (ask) -eIF2: binds to the small ribosomal complex and forms the pre initiation couples -that goes and binds to the cap (eIF-4E) -starts to slide down the mRNA looking for the start codon -when it ﬁnds it the large ribosomal subunit binds and begins making proteins -remember steps of this!!! -energy for protein synthesis comes from GTP -Initiation, elongation, termination Termination -the last bond between the amino acid on the polypeptide and tRNA gets broken by hydrolysis These drugs only bind to the bacterial ribosome, bacteria reproduces so fast that the active sites change and become resistant to the antibiotics -do know the targets of all the drugs How does a cell know that a protein needs to be degraded, it tags it with polyubiquitin -then it is dragged over to the proteasome, like a hollow barrel and inside there are proteases that chew up the protein -ubitiquin tag is not degraded, and recycled -this is important in the cell cycle because not only bad proteins are degraded but proteins that are not needed at the time Examples of gene regulation -All cells contain all the genes in the genome -different cells in the body and turn on different genes -taking a skin cells and turning into the embryonic cell -in diseases you want to determine which genes are on and off in a cell Microarray is divided into sections and each one has a particular square in it -each square you glue mRNA from different genes -then you take a cell and break the cell and extract RNA and you let the RNA hybridize to the microarray mRNA, and the square will glow showing you which mRNA is being produced in the cell -also can see the volume of those mRNA by the intensity of the glow Even gene expression control occurs in the protein activity control DNA binding proteins -helix-turn-helix -zinc ﬁngers -luecine zippers -all bind to the major groove of DNA -they help RNA polymerase bind to DNA Chumley’s material DNA binding motifs (shapes) -they can activate gene expression or deactivate, make it easier for transcription factors to bind, they increase the rate of initiation of transcription (increase the basal level activity) Helix-turn-helix- bind to the major groove, they recognize the sequence because of the temporary bonds made between amino acids on protein and the bases of the DNA, because of this it is very speciﬁc, and lots of weak bonds are kinda strong Zinc Finger- have a zinc atom, looks like ﬁnger Leucine- two polypeptides comes together when leucine binds, cooperative binding, looks like a zipper Dimer formation can determine eh activity of a DNA binding protein -you need two domains to have the dimer bind to the DNA -when they are the same = homodimer -different = heterodimer Ex: MyoD = gene differentiation protein that makes a cell function like a muscle cell, if you take it and put it in a skin cell, it will become a muscle cell Positive/ negative control: The Lac Operon Operon- group of genes that turn on and off a gene?; an efﬁcient way to make the genes that are required to make one protein Lac operon- digest lactose and get energy from it -it takes less energy to break down glucose -if you feed the bacteria glucose and lactose they will reject the lactose When bacteria is given Glucose/Lactose: operon off Glucose: shut down the operon, make sure it doesn’t accidentally come on, a repressor Neither: cap can now bind to operon -when there is no glucose in the cell, cAMP levels go up and vise versa -cAMP is a cofactor, binds to cap and makes it bind to the regulatory sequence Lactose: operon on, removes repressor, need to speciﬁcally not have glucose **know functions of everything, know how its activated and deactivated Enhanceosomes -eukaryotic from of gene regulation that includes dozens of transcription regulators, they bring different parts of DNA together and make an enhanceosome, made up of a lot of little enhancers -there certain proteins that bend the DNA -Cytosine genes- your body makes proteins when this is turned on that make you feel crappy like when you have a cold Combinatorial Control -coactivators can come together in different combinations and still be very speciﬁc for various genes Another way to regulate gene expression Modify the DNA -methylation of DNA can regulate gene expression by silencing promoters -puts a methyl on the cytosines, not every single one -methylation patterns can be inherited by daughter cells or offspring -(epigenetic control- not changing the gene itself but rather the modiﬁcation pattern) -examples: vitamin b, folic acid, BPA, smoking, malnourishent, temperature in the embryo, licking your pups 2 Mice getting fed BPA but one mother was fed supplements with choline, folic aid, betaine -the one that was given BPA plus a normal diet turned out to have a pup that was super fat and abnormal -mother who got a supplements along with BPA had normal baby RNA interference post-transcriptional regulation of gene expression -degredation of the RNA -done in a speciﬁc manner -microRNA is short RNA that folds on itself to make a double stranded structure -and they are complementary to similar RNA -formation of RISC -knock down DNA expression by preventing RNA expression by degrading the mRNA Drosophila embryonic development Ephing Colon Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development Mouse: penis and anus fuse together because of a protein during development Proteins in the membrane -adhere to each other (cell to cell) -their purpose is to send signals to one side of the cell to the other Lipids that make up the membrane -hydrophobic part -hydrophilic part -The difference between major mammalian phospholipids are the head groups Membranes: Functions 1. boundaries of cells are formed by biological membranes -they provide barriers that deﬁne the inside and outside of cells and intracellular organelles 2. They consist of transport systems (proteins) that allow speciﬁc molecules to be taken up and unwanted compounds to be removed from cells 3. Contain components essential in communicating with extracellular world Features -membranes are sheetlike structures, only two molecule thick having dimensions in the range 50-100 angstroms -Consist mainly of lipids and proteins and some carbohydrates linked to lipids Membrane lipids three types: phospholipids -have 4 components -fatty acid tail -platform to which fatty acids are attached (usually glycerol) -fatty acids: hydrocarbons that end with carboxylic group -a phosphate group -an alcohol attached to phosphate group -they all have a hydrophilic head and a hydrophobic tail -so the whole thing is polar -they thing that changes in various phospholipids is the head group (alcohol group) Bilayers in water -the phospholipids aggregate together -when something is hydrophobic, water doesn’t want to interact with it so it forms a cage- like structure -this structure is NOT very energetically favorable, it is an increase in free energy -free fatty acids, has only one fatty acid tail -so their formation is a micelle, the single hydrophobic tail is pointed towards the middle of the ball -Phospholipids -two fatty acids tails, they fo
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