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Exam 2 Study Guide

by: Jesseba Fernando

Exam 2 Study Guide MCB 3010

Jesseba Fernando

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These notes cover the topics you will see on Exam 2
Dr. Landin
Study Guide
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This 41 page Study Guide was uploaded by Jesseba Fernando on Tuesday February 16, 2016. The Study Guide belongs to MCB 3010 at University of Connecticut taught by Dr. Landin in Winter 2016. Since its upload, it has received 48 views. For similar materials see Biochemistry in Microbiology at University of Connecticut.

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Date Created: 02/16/16
Study Guide for Exam II Chapter 7.1, 7.2 and a bit of 7.3 Carbohydrates Functions of carbohydrates  Energy source and energy storage  Structural component of cell walls and exoskeleton  Informational molecules in cell-cell signaling Naming monosaccharides Aldehydes vs ketones  Aldose: contains aldehyde group  Ketose: contains ketone group Triose, tetrose, pentose, hexose  Triose: 3C  Tetrose: 4C o 4 stereoisomers of aldotetroses  Pentose: 5C  Hexose: 6C  Each additional carbon multiplies number of stereoisomers by a factor of 2 o 4 aldotetroses, 8 aldopentoses, 16 aldohexoses Pyranose vs furanose  ribose = furanose, 5C ring o o phosphorylated at C5--- ribofuranose in nucleotides and nucleic acids o attachement of base is in beta config o deoxyribose has 2H at C2  glucose = pyranose, 6C ring o o fructose is ketose form of glucose  Cyclization of C4 leads to furanose o o C1 and C6 not in the ring   Cyclization of C5(penultimate C) leads to pyranose Hemiacetal, hemiketal, acetal, ketal and how they relate to structure of carbohydrates  Aldehydes and ketone carbons are electrophilic  Alcohol oxygen atoms are nucleophilic  Aldehydes attacked by alcohol = hemiacetal  Ketone attacked by alcohol = hemiketal Enantiomers, diasteriomers, epimers, anomers  D-Erythrose and D-Threose are diastereomers  D-Erythrose and L-Erythrose are enentiomers  Epimers: 2 sugars that differ in config around 1 carbon o D-Mannose and D-Glucose  Carbon 2 o D-Glucose and D-Galactose  Carbon 4 Draw Fischer and Halworth projections of glucose, fructose, and ribose Understand L vs D  Hydroxyl group on penultimate carbon o Left on fisher: L o Right on fisher: D Understand α vs β  Beta config o When OH on anomeric carbon is on same side of ring as the CH2OH on Carbon 6  Cis  Alpha config o When OH on anomeric carbon is on opp. Side of ring as CH2OH on Carbon 6  Trans  OH group on anomeric carbon points down on a-D-sugars and b-L- sugars. It points up for b-D-sugars and a-L-sugars o Anomeric config (a/b) is the same when monosaccardide is mirror imaged  a-D-glucopyranose is a-L-glucopyranose Chair conformation  Pyranoses exist in chair conformation o Glucose: all hydroxyls are equatorial except at C1 in alpha form o Two possible chair conformations- bulky substituents go in equatorial for stability Disaccharides-name monosaccharides and their binding for Maltose, Lactose, and Sucrose  Formation of glycosidic bonds between monosaccharides = disaccharide  Alpha Hemiacetal + beta alcohol (cis) ->condensation-> (<-hydrolysis<-) alpha-disaccharide  Beta hemiacetal + beta alcohol (trans) ->condensation-> beta lactose  Lactose: galactopyranosyl + glucopyranose  Sucrose: fructofuranosyl + glucopyranoside  Trehalose: glucopyranosyl + glucopyranoside Polysaccharides  Natural carbs found as polymers o Can be linear, branched, homo or hetero, no defined MW, no template used to make polysaccharides General structure (monosaccharides, branching, connections) and function of glycogen, starch, cellulose o Glycogen  Branched homopolysaccharide of glucose  Alpha 1-> 4 chain  Branch points alpha 1 -> 6  MW reaches several millions  Main function: storage in animals o Starch  Mixture of two homopolysaccharides of glucose  Amylose is unbranched polymer of (alpha 1 -> 4) linked residues  Amylopectin is branched polymer, branch points at alpha 1->6 occurring every 24-30 residues  MW is up to 200 million daltons  Main function: storage in plants o Cellulose  Unbranched homopolysaccharide of glucose  Glucose monomers form (beta 1->4) linked chains  Hydrogen bonds beterween adjacent monomers  Additional H bonds between chains  Tough and water insoluble  Most abundant in nature  Structure element in plants-wood, cotton  Metabolism  Fibrous structure and water insolubility = difficult substrate to act on  Fungi, bacteria, protozoa secrete cellulose o Use wood as source of glucose  Most animals cannot use as fuel source o Lack enzyme to hydrolyze beta 1->4 linkages  Ruminants and termites live symbiotically with microorganisms that produce cellulose  Promise of fermentation of biomass in fuels  Chitin  Major building block of exoskeleton of arthropods and some fungi  Structure is identical to cellulose except glucose is replaced by N-acetylglucosamine  Tough, flexible, water in-soluble o Tertiary Structure  Weak interactions, rigid structural areas, steric hindrance, limited bonds  Limited rotation  Start and glycogen = tightly coiled helix stabilized H bonding  Cellulose: extended structure with H bonding between chains Glycoproteins-amino acids that bind carbohydrates, function of carbohydrates  Oligosaccharides attached to proteins at anomeric carbon to oxygen of Ser or Thr residues (O-linked) or nitrogen of Asn (N-linked)  ½ all protein glycosylated  increases solubility, can target proteins for certain cellular locations, can change structure Chapter 8 Nucleic Acids Functions of nucleotides (starts and ends the slides) and nucleic acids  Nucleotides o Energy for metabolism (ATP) o Enzyme cofactors (NAD+) o Signal Transduction (cAMP)  Nucleic Acids o Storage of genetic info (DNA) o Transmission of genetic info (mRNA) o Processing of genetic info (ribozymes) o Protein synthesis (tRNA, rRNA) Nucleotides vs nucleosides  Nucleotide o Nitrogenous base, C1 beta o Pentose sugar o Phosphate, C5  Negatively charged at neutral pH  At 5’ position of pentose, C5  Buil using 5’ triphosphated (ATP, GTP, TTP, CTP)  One per nucleotide  Can also be 5’ diphosphates (ADP, GDP, TDP, CDP)  Can be attached to other positions o Nitrogenous base o Pentose  Nucleobase o Nitrogenous base Draw nucleotide if structure of nitrogenous base provided  Slide 5 Pyrimidines and purines  Slide 3  Nitrogen containing heteroaromatic molecules  Planar/almost planar  Pyrimidine o Cytosine: both DNA and RNA o Thymine: DNA o Uracil: RNA o Good H bond donors/acceptors, neutral at pH 7 o Slide 9  Purine o Adenine, guanine found in both RNA and DNA o Good H bond donor/acceptor o Neutral molecules at pH 7 C-2’endo and C-3’ endo, syn and anti as pertains to forms of DNA  b-D-ribofuranose in RNA  b-2’-deoxy-D-ribofuranose in DNA  diff. puckered conformations in sugar rings  Slides 6-7  Free rotation in free nt’s  Torsion angle N-glucosidic bond (N-C1) is denoted by chi (X)  Angle near 0 is syn conformation  Angle near 190 is anti conformation  Anti conformation found in normal B-DNA UV absorbance of nucleotides/DNA/RNA  Absorption of UV light at 250-270 nm due to pi -> pi* electronic transitions  Molar extinction coefficient o AMP: 15,400 o GMP: 11700 o UMP: 9,900 o dTMP: 9,200 o CMP: 7,500 Differences between DNA and RNA, including which carbon loses OH  Slide 16, 17  C2 Role of Methylation  Modification done after DNA synthesis o 5-Methylcytosine in euk is common, also found in bacteria o N6-Methyladenosine is common in bacteria, none in euk  Epigenetic marker o Way to mark own DNA so that cells can degrade foreign DNA -prokaryotes o Ways to mark which genes should be active  Methylated genes not transcribed- euk Structure of DNA and RNA How backbone is linked together  Covalent bonds formed via phosphodiester linkages o Neg. charged backbone  DNA Fairly stable o DNA from mammoths o Hydrolysis accelerated by enzymes- DNAse  RNA backbone unstable o In water, RNA lasts for a few years o In cells, mRNA is degraded in few hours  Linear o No branching, poylmers  Directionality o Read the seq from 5’ to 3’ Base pairing  B-N-glycosidic bond o Forms to anomeric carbon of the sugar in B config o N1 position in pyrimidines o N9 in purines o Stable toward hydrolysis 5’ and 3’ ends  Slide 21 Hydrogen bonding and base stacking hold structures together  2 bases + H bonding = base pair  A = T, C Ξ G  Purine – pyrimidine pairings o 2 purines too far apart, 2 pyrimidines too close  in RNA, sometimes G pairs with U but only for structure formation/anticodon wobble  more energy to break G-C bonds Differences between DNA and RNA DNA-double helix, overall structure, general structural differences between A, B, and Z forms (don’t need to memorize numbers ie nucleotides per turn for each one)  Double Helix o Frankin and Wilkins  Cross means helix  Diamonds mean that the phosphate sugar backbone is outside  Helical parameters calculated o Watson and Crick  Missing layer in the cross means alternating pattern  Major and minor groove  H bonding  A with T  C with G  Double helix!!  Complementarity of DNA Strands o Read from 5’ to 3’ o Antiparallel o Stabilized by H bonding and base stacking  B DNA o Major groove: 36Å o Minor groove: 3.4Å o Right hand twist o Diameter: 20Å o Bp per helical turn: 10.5 o Helix rise per bp: 3.4Å o Base tilt normal to helix axis: 6 degrees o Sugar pucker: C-2’ endo o Glycosyl bond conformation: anti  A DNA o Right handed o Diameter: 26Å o Bp per helical turn: 11 o Helix rise per bp: 2.6Å o Base tilt normal to helix axis: 20 degrees o Sugar pucker: C-3’ endo o Glycosyl bond conformation: anti  Z DNA o Left handed o Diameter: 18Å o Bp per helical turn: 12 o Helix rise per bp: 3.7Å o Base tilt normal to helix axis: 7 degrees o Sugar pucker: C-2’ endo for pyrimidines, C-3’ endo for purines o Glycosyl bond conformation: anti for pyrimidines, syn for purines RNA-formation of complex structures, palindromes, role of non-covalent interactions in structure  mRNA o code carrier for the sequence of proteins o synthesized using DNA template o contains ribose instead of deoxyribose o contains uracil instead of thymine o ss instead of ds o 1 mRNA codes for more than 1 protein o mRNA + tRNA transfers genetic information from DNA to make proteins  Monocistronic: 1 gene in 1 strand of RNA  Polycistonic: multiple genes in one strand of RNA  Palindromic sequences o Words/phrases that are the same read forwards and backwards o These RNA sequences can form hairpins and cruciform  Base stacking- non-covalent interaction between planar ring moieties DNA denaturation- causes and what happens structurally- what affects temperature at which DNA melts  Covalent bonds remain intact thereby genetic code is intact  H bonds broken o Strands separate  Base stacking is lost  UV absorbance increases  High temp and pH change o Exists as double helix at normal temp o Strands dissociate at elevated temp o Re-anneal when temp is lowered o Reversible thermal denaturation/annealing is basis for PCR o Monitored at UV spectrophotometry at 260 nm  Factors o Midpoint of melting (Tm) depends on base composition  High GC content increases Tm  Harder to break H bonds o DNA length  Longer DNA has higher Tm  Important for short DNA! o pH and ionic strength  high salt increases Tm  Can be reversible: annealing Mutation-types and what happens to the nucleotide structure, environmental causes of mutations  Two near complementary DNA strands can hybridize o Detection of DNA molecule in mixture  Radioactive detection  Fluorescent DNA chips o Amplification of DNA  PCR  Site directed mutagenesis o Shows evolutionary relationships  Functions of nucleotides o Energy source o Coenzymes o Regulatory molecules Chapter 10.1-10.2 Lipids Functions of lipids- advantages for energy storage  Energy storage o Reduced compounds-lots of energy available o Hydrophobic nature: good packing  Insulation o Low thermal conductivity o High heat capacity (absorbs heat) o Mechanical protection  Water repellant o Hydrophobic, keeps surface dry o Precents excessive wetting o Prevents water loss via evaporation  Bouyancy control/acoustics o Increases density while diving deep helps sinking o Spermaceti organ may focus sound energy  Sound stun gun?  Membrane structure  Cofactors for enzymes o Vitamin K—blood clot formation o Coenzyme Q: ATP synthesis  Signalling molecules o Paracrine- local o Steroid- body wide o Growth o Vit A and D (hormone precursor)  Pigments o Tomatoes, carrots, pumpkins and birds  Antioxidants o Vit E Fatty Acids- draw structure  7-6  4-36 carbons (mostly even number)  unbranches (natural ones) naming using x:y (Δ ) and meaning of omega-3 and -6  cis-9-octadecenoic acid o 18:1(delta^9) o 18 carbons o 1 double bond  at Carbon 9  w naming starts counting carbons the furthest from COO- Concept of saturated, monounsaturated, polyunsaturated  saturated = no double bonds (fully saturated with hydrogen)  monounsaturated = one double bond in alkyl chain  polyunsaturated: more than one double bond  conformation o double bonds are common in cis  kinks the chain How structure affects solubility and melting point and reasoning for those effects  solubility o decreases as chain length increases  MP o Increases as chain length increases o Decreases as the # of double bonds increase  Saturated fatty acids pack in fairly orderly way  Extensive favorable reactions  Unsaturated cis fatty acid—less orderly bc of kink  Less extensive favorable interactions  LESS THERMAL ENERGY TO DIRUPT DISORDER Trans fatty acids-trans isomer around double bond, like saturated fatty acid  Form by partial hydrogenation of unsaturated fatty acids o Done to increase shelf life/stability at high temp o Produced by bacteria in ruminant stomach  Trans double bond allows fatty acid to adopt extended conformat like saturated fatty acid Triacyl glycerol-draw structure, why more hydrophobic than fatty acids alone,main storage molecule  Three fatty acids + ester linkage + 3 OH groups of glycerol  Majority of fatty acids found in form of triglycerols  Solid = fat  Liquid = oil  Primary storage  Less soluble in water than fatty acid (lack of charged hydroxyl)  Less dense than water: fats and oils float  Slide 15 Waxes- describe general structure and function  Esters of long chains saturated/unsaturated fatty acids with long chain alcohol  Insoluble, high melting point  Functions o Storage of metabolic fuel in plankton o Protection/pliability for hair and skin o Waterproofing feathers o Protection from evaporation in plants o Used by people in lotion/ointment, polish  Slide 17 Glycerophospholipids- describe general structure and function, orientation in membranes  Lipids in memebranes o Polar head, nonpolar tail  Diversificaition--- diff backbone, diff fatty acid, add head groups  Prop of head groups determine surface prop. o Diff organisms have diff membrane lipid comp. o Diff. tissues or diff. memebranes within a cell have diff. membrane lipid comp.  Glycerophospholipid o Primary constituent for cell membrane o Two fatty acid fors ester linkages with 1 and 2 ndhydroxyl groups of L-glycerol-3-phosphate o Head group is charged at physiological pH o General structure- slide 20  Unsaturated fatty acid connected to C2  Highly polar phosphate group esterified by alcogol  Head group  Examples: slide 21 o E.coli cannot synth. Lipids so do not have phosphotidylcholine Sphingolipids-describe general structure and function  Backbone: not glycerol o Sphingosine  Fatty acid joined through maide linkage  Polar head connected by glycosidic or phosphodiester bond o Sugar containing glycosphingolipid found on outer plasma membrane  Structure: slide 24 Sterols-describe general structure and function  Steroid nucleus o 4 fused rings  hydroxyl group (polar head) in A ring  steroid nucleus is almost planar  Function o Cholesterol and sterols  Most euk cell membranes  Fluidity, permeability  Thickness  Bacteria lack sterols o Mammals obstian from food or synthesize in liver o Transported to tissues in lipoproteins  Combo of lipid and proteins  Densirt small particles have high density  Cholesterol- low desnity lipoproteins o Hormones are derivatives of sterols Fats over polysaccs  Fatty acids = more energy per carbon—more reduced  Carry less water bc nonpolar  Glucose/glycogen for short term energy  Fats are long term/months—slow delivery Chapter 9.1 and 9.3 DNA Techniques Agarose gel electrophoresis-how it works, uses  Neg. charged DNA migrates to anode in presence of electric field  Agarose gel hinder motility o Depends on size and shape  Small molecules travel faster  Compact molecules travel faster  DNA detected by dyes which sit between base pairs and fluoresce under UV light  Uses o DNA analysis, for size or purity o DNA purification o DNA-protein interaction studies PCR-how it works and what is achieved  Create mixture o Target DNA o Primers (oligonucleotides complementary to target) o Nucleotides, dATP, dCTP, dGTP, dTTP o Thermostable DNA polymerase  Synthesizes DNA  Place micture into thermocycler o Melt DNA at 95C o Cool separated strands to 50-60C o Primers anneal target o Polymerase extends primers in 5’-3’ direction  Makes complementary strand o Repeat  Uses: o Amplify DNA in a tube  Can amplify regions of interest within linear DNA  Can amplify complete circular plasmid Sequencing-from Chapter 8- how it works, what is determined  Step 1: run PCR reaction o With DNA t be sequenced as target o Primers o dNTPs, and ddNTPs  ddNTPs block elongation and are tagged with fluorescent group uique to each ddNTP (lack C3’ hydroxyl  Step 2: run sample through capillary o Electrophoresis to separate out DNA strands based on sixe  ddNTP fluorescence detected  Uses: o Verify gene of interest via correct sequence o Determine seq of gene o Determine mutation in gene  Types: o Full Genome  Immobilized on chip in fragments  Sequenced at once o Pyrosequencing (454)  Synthesizes DNA from template  Single nt at a time  Pulse of light when each added  Read 400-500 nts in the sequence o Reversible terminator sequencing  Fluorescently labeld terminal nt added/detected  Terminal blocker/fluorescent label is removed, seq extended, next nt detected DNA cloning-how it works and use  Recombinant DNA o Artificially created DNA- combines seq’s that don’t happen naturally o Basis of modern MCB  Molecular cloning of genes  Over-expression of proteins  Transgenic animals o Restriction Endonucleases  Cleave DNA phosphodiester bonds at specific seq.  Common in bacteria  Eliminates viral DNA  Can make staggered cuts (EcoRI)  Sticky ends  Straight cuts (PvuII)  Blunt ends o DNA Ligase  Covalently joins two DNA fragments  Normally in DNA repair  Human DNA ligase uses ATP  Bacterial ligase uses NAD  DNA Cloning o Creation of identical copies of a piece of DNA/gene  Isolate specific gene from source organism an amplify it in target organism (bacteria) o Steps:  Cut source DNA at boundaries/use PCR to copy it from source DNA  Select carrier DNA (vector)  Insert gene into vector  Insert recombinant vector into host cell  Let the host produce multiple copies of recombinant DNA Differences between cloning and expression vectors  Cloning vectors o Plasmid: circular DNA molecules separate from bacterial genomic DNA  Replicates autonomously  Origins of replication used for bacteria/yeast  Carry antibiotic resistant genes  Allows cloning of DNA up to 15,000 bp  High copy number of vectors o Clone entire chromosome  BAC  For bacteria  YAC  For yeast o Viruses: express proteins in insects and mammalian cells o Transformation  Need plasmid with new gene into cell- usually bacterium  Heat shock- make cells competent first  Electroporation: cells take up DNA o Antibiotic selection  Kanamycin and amp kill bacteria  Plasmids carry genes that give host bacterium resistance against antibiotics  Only allows growth of bacteria that have taken up the plasmid  Expression Vectors o Use: study protein product of a gene o Special plasmids  Contain sequences that allow transcription of the inserted gene-  Usually low copy number plasmids o Expression vectors differ from cloning vectors by having  Promoter sequences  Operator sequences  Code for ribosome binding site  Transcription termination sequences o Use expression bacterial cells o Purification of recombinant proteins  Of natural proteins is hard  Can be tagged for purification  Binds to the affinity resin, binding the protein of interest to a purification column DNA fingerprinting- what is determined, how is it determined, what guarantees uniqueness of the results from a particular person, CODIS  Humans have short sequences repeats next to each other (STRs)  Differences in number of repeats cause variations in length of fragments  Fragment sizes determined by using capillary electrophoresis  Multiple STR locations exist in human genome  Allows matching suspect samples to known individuals  13 well studied locations used for identifications o based on number of alleles, misidentification is less than 1 in 10^18  CODIS is a database with repeat motifs Single Nucleotide polymorphisms-what they are  Distinguish human populations  Linkage of SNPs to disease inheritance can identify genes involves New sequencing techniques- differences between new techniques and normal sequencing  Human Genome Project o 1990 to 2003 o identification and categorization of genetic differences between individuals o understanding the differences between human and other organisms  based on phylogenetic trees and comparison of differences  especially in regulatory sequences, which may be more important to evolution than protein changes o identification of genes involved in disease o track the path of human migration  genome contains many diff sequence types o simple seq repeats o segment duplications o long repetitive seq, centromere/telomere o misc o transposons o SINEs o LINEs o Protein coding ~ 1.5% Chapter 25.1-25.2 DNA Metabolism (Replication and Repair) Exonucleases and Endonucleases-function, differences between them  Exonucleases: cleave bonds that remove nucleotides from the ends of DNA  Endonucleases: cleave bonds within a DNA sequence  Usually fix for errors/degrade foreign DNA Purpose of Replication  Begins at origin, proceeds bidirectionally  Synthesis of new DNA occurs 5’->3’ direction, semi-discontinuous Replication Semi-conservative  DNA strands are separated and a new strand is synthesized that is complementary to original strand  Messelson-Stahl Experiment o Proved Watson-Crick hypothesis o Cells grown in media with 15N isotope (heavy N)  DNA fully labeled with 15N  One band when centrifuged in CsCl gradient o Then cells switched to 14N medium allowed to divide once  One band but at higher position than 15N but lower than 14N  Hybrid!! o Cells allowed to divide once more  Two bands!  14N DNA and 1 hybrid Proteins involved-functions of proteins, active site and catalytic action of polymerases, different functionalities of polymerases including direction of action along DNA  DNA pol I discovered in E.coli, which has at least 4 other DNA polymerases o Synthesizes DNA  Parent strand serves as template  Nucleoside triphosphates are substrates  Nucleophilic OH group at 3’end attacks alpha-phosphate (nearest to deoxyribose) of incoming trinucleotide o 3’OH REQUIRED o 3’OH made more powerful by nearby magnesium ions  pyrophosphate (beta and gamma) is a good leaving group  DNA pol requires a primer o Short strand complementary to template  Has 3’OH to brgin DNA pol catalyzed reaction  Made of RNA  Mechanism: o Slide 15, DNA rep  Features o Has pocket with 2 regions  Insertion side: incoming nt binds  Post-insertion site: newly made bp resides when polymerase moves forward  Geometry of base pairing help high fidelity o Errors in E.coli 1 per 1000 to 10,000 o DNA pol active site excludes base pairs with incorrect geometry  But still insert wrong base 1/10^4 to 1/10^5 times o Repair mechanisms fix these Differences between leading and lagging strand synthesis  synthesis is always adding nt’s to the 3’ OH  leading strand is continuous, lagging is discontinuous (okazaki frags) Steps of Replication in prokaryotes-Initiation, Elongation, Temination- what is performed at each step and what proteins are involved  E.coli requires over 20 proteins for replication o “replisome”  Helicase: uses ATP to unwind DNA strand  Topoisomerase: relieve the stress caused by unwinding  DNA-binding proteins: to stabilize separated strands  Primases: to make RNA primers 5-15 bases long  DNA ligases: to seal nicks  Initiation o Begins at OriC—245 bp long  Highly conserved sequence  9 bp repeated 5 times- R sites  binding site for insitiator protein (DnaA)  contains AT rich DNA unwinding element (DUE)  more sites for DnaA (I sites) o 8 DnaA proteins bind at and I sites in OriC o DnaA proteins = ATPases o DNA wraps around the complex forming positive supercoil o Strain leads to denaturation of nearby DNA Unwinding Element (DUE) site o Associated proteins facilitate DNA bending  IHF- integration host factor  FIS- factor for inversion stimulation  DnaB (helicase) binds and migrates along ssDNA 5’- >3’ (on one strand) and unwinds the helix  Other proteins (DNA pol II link to DnaB  ss DNA binding protein stabilizes separated strands  DNA gyrase (topoisomerase) relieves topological stress ahead of the replication forks  Elongation o Leading strand synthesis  Primase (DnaG) makes RNA primer  Primase interacts with DnaB helicase  Primer made in 5’->3’  DNA pol III adds deoxynucleotides in the 5’->3’ direction starting at end of primer o Lagging strand synthesis  Primase makes RNA primer, and DNA pol III adds nucleotides in 5’>3’ direction  Except in short segments! (ends when it hits previous primer)  Same DNA pol III works on both strands  The DNA of lagging strand loops around- slide 39 to 42  DNA pol I removes NMPs and dNTPs added in  DNA ligase uses ATP to seal nicks  5’-PO4 is activated by attachment of AMP  3’OH nucleophile attacks this phosphate, displacing AMP  slide 46  Termination o Replication forks meet at region with 20 bp sequences Ter (TerA-TerF)  Ter sites found near each other but in opp directions  Create a site that replication forks cannot leave o Ter is binding site for protein Tus  Tus = terminus utilization substance  Causes a replication fork to stop  In eukaryotes o More complex o ORC loads helicase onto DNA  ORC functions like DnaA  Helicase is a hexamer of mini chromosome maintenance proteins MCM2-7  Functions like DnaB helicase o Replication is slow but from many origins th  ~50 nt per sec (1/20 the rate of e.coli)  termination occurs when replication forks converge  telomeres allow ends of chromosomes to be replicated by providing a place for the primer to bind o DNA polymerases  DNA Pol alpha  Primers with both RNA and DNA  No 3’->5’ proofreading  DNA Pol delta  DNA synthesis  Comparable to DNA pol III  Maybe lagging strand only  Has 3’->5’ proofreading  Involved in repair  DNA pol sigma  Similar to delta  Prob just on leading strand How errors are corrected  errors during synthesis corrected by 3’-5’ Exonuclease activity o most DNA pol have additional activity o 3’->5’ Exonuclease activity proofreads synthesis for mismatched bp o translocation of enzyme to next position is inhibited until enzyme can remove the incorrect nucleotide How primer is added and replaced at the end  Primer is added by Primase  Removal: o Core subunits of DNA pol III dissociate from one beta clamp, bind to new one o RNA primer is removed by DNA pol I o DNA polymerase I fills in the gap o DNA ligase seals the nick o RNA primer must also be placed on leading strand Differences between eukaryotic and prokaryotic replication  E.coli o At least 5 DNA pol o DNA pol I  Abundant, not ideal for replication  Rate 600 nt/min—slower than observed  Low processivity (falls of DNA frequently)  Primary function is clean up  Has 5’->3’ Exonuclease activity  Moves ahead of enzyme- hydrolyzes thing in its path  Nick translation- strand break moves along enzyme  Distinct domain: Klenow fragment o Domains can be separated by protease treatment o DNA pol III  Principal replication polymerase  Over 10 subunits  Two core domains of alpha, epsilon, and theta subunits  Core domains linked by “clamp loader compelx” t1ydd  Core domains interact with dimer of beta subunits- increases processivity o Sliding clamp prevents dissociation o Processivity of DNA pol II is >500,000 bp  Has 3’->5’ Exonuclease proofreading  No 5’->3’ exonuclease o DNA pol II, IV, V are involved in DNA repair  DNA pol II  Also does not have 5’->3’ Exonuclease  7 subunits  Repair  chemical reactions and physical processes damage genomic DNA o majority are corrected using the undamaged strand as template o sometime bases aren’t repaired and serve as templates in replication o daughter DNA carries changed sequence in both strands o mutations correlated with cancer For each repair mechanism (Mismatch, Base Excision, Nucleotide Excision):  Mismatch o Arise from occasional incorporation of incorrect nuvleotides o Repair relies on methylation  Repair enzymes know the correct strand  E.coli, parent strand is methylated  Dam methylase inserts CH3 on adenines in GATC seq  Following short period of time after replication, daughter strand is methylated  Methyl directed mismatch repair system will cleave the unmethylated strand in initial part of repair  Cleaved strand is degraded by exonucleases and rebuilt by DNA pol  MutL and Mut S bind at mismatch using ATP  MutH binds to MutL-MutS complex making DNA loop o Includes methylated ATC o Mismatch can be more than 1000 bp away from GATC  MutH cleaves nonmethylated DNA strand on 5’ side  DNA unwinds and is degraded  Helicase II and exonucleas work to degrade non- methylated DNA toward mismatch  Removed sequence is replaced usng DNA pol III and DNA ligase  Very energy intensive-removing and resynthesizing many nucleotides just to repair one (in E.coli)  Abnormal Bases o Arise from spontaneous deamination, chemical alkylation or exposure to free radicals o Base excision repair uses DNA glycosylases  Used to remove deaminated NMP along with oxidized or alkylated NMP  DNA glycosylases recognize specific lesions  Cleave N-glycosyl bond between sugar and base o Creates apurinic/apyrimidic AP site  Uracil glycosylase removes uracil from DNA o Important bc C spontaneously deaminates to U  U doesn’t belong in DNA  Entire nt is removed not just damaged base  Sometimes region around AP site removed  AP endonuclease cuts the DNA backbone 5’ of AP site  DNA pol I removes DNA with 5’->3’ Exonuclease activity  Synthesizes new DNA  DNA ligase seals the nick  Pyrimidine dimers o Form when DNA is exposed to UV light o Large distortions in DNA are repaired by nucleotide excision  Ie: pyrimidine dimers and 6.4-photoproducts o Pathway involves removal of a DNA segment by excinucleases  Excinucleases cleave DNA backbone in two places  ABC excinuclease  Hydrolyzes 5 bond on 3’ side of lesion and 8 th bond on 5’ side  Removes 12-13 nucleotides  DNA pol I and DNA ligase replace the DNA and seal the gap (on 3’side)  Backbone Lesions o Exposure to ionizing radiation, free radicals Chapter 26.1 and 26.2 RNA Metabolism (Transcription and Eukaryotic RNA Processing) Transcription  In E.coli o Nucleoside triphosphates added to 3’ end of RNA strand o Complementary to template strand in DNA o Catalyzed by RNA polymerase o No need for primer  First nucleotide retains all three phosphates on 5’ end  Slide5 picture RNA polymerase-active site, catalytic mechanism, other activities, lack of proofreading  RNA pol binds to promoter sequence to begin transcription o Primer not required  Growing end of RNA temporarily base pairs with DNA template for ~8bp  DNA duplex unwinds, formin bubble about ~17 bp  RNA pol generates positive supercoils ahead, relieved by topoisomerases  Slide 7 picture  Multi-subunit protein o Holoenzyme: has 5 core subunits  Alpha2betabeta’w, plus sixth called sigma  2Alpha subunits: function in assembly, binding to upstream promoter elements  beta subunit is main catalytic subunit  beta’ is responsible for DNA binding  sigma subunit directs enzyme to the promoter  w appears to protect polymerase from denaturation o lacks 3’->5’ Exonuclease  has high error rate of 1/10^4 to 1/10^5 nts Role and elements of promoters  Promoters in E.coli that bind the same RNA pol have common features o 2 consensus sequences at -10 (TATAAT): TATA box and -35 (TTGACA) for sigma subunit binding  TATA sequences o AT rich upstream promoter elements between -40 and -60 binds alpha subunit o AT rich sequences promote strand separation  2 H bonds o Sequences govern efficacy of RNA pol binding  affect gene expression level Role of σ subunits in transcription  sigma70: housekeeping o slide 16  sigma54: moderates cellular nitrogen levels  sigma38: stationary phase genes  sigma32: heat shock genes o slide 16  sigma28: flagella and chemotaxis genes  sigma24: extracytoplasmic functions, some heat shock  sigma18: extracytoplasmic functions: ferrc citrate transport Steps of transcription in prokaryotes-initiation, elongation and termination-what happens at each step, proteins involved  RNA pol core binds to DNA promoter  Closed complex  Transcription bubble forms from -10 to +2  RNA pol moves away from promoter o Sigma dissociates allowing elongation  Sigma 70 dissociates and is replaced by NusA  Transcription terminated. NusA dissociates and RNA pol is recycled o P-independent termination in E.coli  Characterized by multiple U’s near 3’ end  Self complementary region in transcript form a hairpin- 15-20 nt before 3’ end  DNA pol pauses  RNA dissociates from DNA o P-dependent termination in E.coli  Known: common CA-rich sequence called a rut site  Rho utilization element  Rho protein is a helicase, binds to rut site (using ATP)  Proceeds 5’ to 3’ until it reaches RNA polymerase paused)—releases the RNA Template and coding strands of DNA-using one or both strands as template strands  Template strand of DNA: template for RNA polymerase  Coding strand of DNA: non-template strand--- same seq as RNA transcript  Both strands may code for proteins o Coding info on either strand  Ex: adenovirus  Causative agents in common cold  Linear genome  Each strand codes for number of proteins DNA footprinting- technique and what the results show  A way to find binding site on DNA for DNA binding protein o DNA bound by protein is protected from chemical cleavage at binding site  Steps o Isolate DNA fragment thought to contain protein binding site o Radiolabel DNA and split sample o Add DNA binding protein to DNA in one tube, other tube is control o Treat both samples with chemical/enzymes to cleave DNA into many pieces o Separate fragments on agarose gel electrophoresis  Visualize bands on X ray film/ imager plate  If protein binds, multiple bands will be missing in the lane for that sample Differences in transcription in eukaryotes and prokaryotes  Eukaryotic transcription o RNA pol I: synthesizes pre-ribosomal RNA o RNA pol II: responsible for synthesis of mRNA  Very fast  Can recognize thousands of promoters o RNA pol III: makes tRNAs and small RNA products o Plants have RNA pol IV: synthesis of small interfering RNAs- siRNAs o Mitochondria have their own RNA polymerase o Involves many proteins  Protein-protein contacts  Highly conserved transcription factors  RNA pol II well studied  Large complex with 12 subunits o Some have structural homology to bacterial RNA polymerase  Carboxyl-terminal domain of highly conserved repeats o Phosphorylated and dephosphorylated during transcription  Proteins: o TBP, TFIIA, B, E, F, H, recruit RNA pol II o Create closed complex o Transcription bubble formsTFIIE, H leave and transcription initates  CTD is phosphorylated during initiation  Polymerase escapes promoter o Elongation factors recruited o Elongation factors dissociate. CTD dephosphorylated as transcription terminates  Facilitated by termination factors  Assembly of RNA pol at promoter o Initiated by TBP: tata binding protein o Helicase activity in TGIIH unwinds DNA o Kinase activity in TFIIH phosphorylates the RNA pol at CTD  Changes conformation- allowing transcription  Elongation and Termination o Elongation factors bound to RNA pol II enhance processivity  Coordinate post-translational modifications o Termination, Pol II is dephosphorylated  May involve mechanism similar to rho-dependent termination in prokaryotes RNA Processing 5’ cap-structure and function  7-methylguanosine links 5’end of mRNA via 5’, 5’-triphosphate link o can include additional methylation of 2’OH groups of next two nucleotides  protects RNA from nucleases  forms binding site for ribosome  cap is added during transcription by 3 enzymes attached to CTD of RNA pol II  slide 34 Removal of introns  exons usually <1000 bp  introns 50-20,000 bp  some genes have dozens of introns  Classes: o Group I and II  Self splicing  Ribozymes o Cleave themselves or another RNA o 3D structure is integral to function o inactive if denatured o Michaelis-Menten kinetics  Saturable, have acive site, heave measurable Km, can be competitively inhibited  Nucleophilic attack of sugar OH on phosphate followed by phosphodiester bond hyrolysis  Nuclear, mitochondrial, and chloroplast  Differ mainly in splicing mechanism o Spliceosomal introns  Spliced by splicesomes containing RNA and proteins  Most common  Frequent in protein-coding regions of euk genomes  tRNA introns  spliced by protein based enzymes  exons are joined by ATP dependent ligase Poly(A) tail- enzyme that adds tail, usefulness  serves as binding site on euk mRNA  after RNA pol II synthesizes TNA, endonuclease cleaves RNA 10- 30 nt downstream from highly conserved AAUAA o free 3’OH  polyadenylate polymerase synthesizes 80-250 nt of A without template How one gene and variable processing of mRNA results in different proteins  single gene can yield diff products dependent on RNA processing o RNA can be edited—bases removed/added o Cleavage of introns/polyadenylation patters vary  Diff. mature transcripts o Calcitonin and calcitonin-gene related peptide  Made from same mRNA—slide 41 Degradation of RNA Limited half life  RNA lifetime one way of gene regulation  Half lives vary from seconds to hours o Average for mRNa ~3hrs o 10 turnovers per cell generation o shorter half-lives for bacterial mRNA (1.5min)  hairpin structures in mRNA can extend half-life Exoribonucleases and Endoribonucleases-both necessary for eukaryotic RNA  Endoribinuclease clips RNA into segments  Exoribonuclease then breaks down fragments or degrades whole RNA strand o Can start at either end  Ribonucleases involved vary Chapter 27-Protein Metabolism (Translation, post-translational modification and degradation) Translation  Uses 90% of chemical energy in cells Genetic code  Written 5’->3’  3 nt for aa o 3 base not that important for tRNA o 1 codon establishes the reading frame  if RF is thrown of by base or two, subsequent codons are out of order  61/64 codon code for aa—degenerate code  termination codons: UAA, UGA, UAG  AUG = initiation/Met  Universal--- few exceptions  Pro and euk use it  Mito has slightly diff code  UGA encodes Trp not STOP  AGA/AGG encodes STOP instead of Arg  Encode their own tRNAS, use 22 instead of 32 o Resistant to mutations  Degenerate code allows for certain mutations bt code for same aa  Silent mutations  Mutation in first base of codon can produce conservative substitution Codons/anticodons-5’to 3’  Codon seq of mRNA is complementary to anticodon seq on tRNA  Codon in mRNA base pairs with anticodon in tRNA via H bonding  Alignment of 2 RNA segments = antiparallel Degeneracy of code-need for 64 codons, advantage of 64 codons Wobble Hypothesis-how it works, relationship to tRNAs  Third base of codon form non-canonical base pairs with its anticodon in tRNA o G can bind U o Some tRNAs have inosinate (I) which can H bond to U, C, and A  H bonds are weaker and called wobble bp o As few as 32 tRNAs to properly translate 61 codons to the correct amino acids--- no tRNAs for STOP o 1 base  C on anticodon recognizes G  A on anticodon recognizes U o 2 bases  U on anticodon recognizes A/G  G on anticodon recognizes C/U o 3 bases  I on anticodon recognizes A/U/C Initiation and Termination codons  AUG- Met/initiation  Termination o In all except mito: UAA, UAG, UGA  No anticodons o Mito: AGA, AGG Activation of amino acids  By binding to tRNA o tRNA is aminoacylated  synthesis o creation of aminoacyl intermediate  aminoacyl tRNA synthetases  Step 1: COO-  aa attacks phosphate of ATP- creates aminoacyladenylate intermediate  pyrophosphate (PPi) is also cleaved, so reaction is driven forward by 2 phosphoanhydride bond cleavages  Slide 27  Transfer of aminocyl to tRNA  Step 2-2’-OH or 3’-OH of tRNA attacks phosphate of aminoacyladenylate intermediate o Ester bond between aa and tRNA  Aa attached to 2’ OH  3’OH attacks phosphate and transfers aa to 3’OH of tRNA  aa now attached to 3’OH of 3’ end of the tRNA  slide 29 Addition of amino acids to tRNA  mRNA and first aminoacylated tRNA bind to ribosome  cycles of aminoacyl tRNA binding and peptide bond formation o until stop codon reached o fMet/Met is first in peptide  AUG  tRNAs for Met  bacteria (chloro and mito)  inserts N-formylmethionine  euk beings with Met not fMet, but special tRNA is still used o Initiation:  30S subunit  mRNA  fMet-tRNA  IF-1, IF-2, IF-3  GTP  50S  Mg2+ Enzyme and steps involved  Aminoacyl-tRNA synthetase o Each enzyme binds a specific aa and the matching tRNA  20 diff aminoacyl-tRNA synthetase (1 for each aa)  must be specific to aa and tRNA o matching each aa with correct tRNA is “second genetic code” o code is molecular recognition Ribsome structure- made of large and small subunit each composed of proteins and rRNA  25% of bacterial dry weight  65% rRNA, 35% protein o rRNA forms the core o RNA does the catalysis of peptide bond formation  Made of 2 subunits o Bacteria: 30S and 50S o Eukaryotes: 40S and 60S o mRNA runs through them  complex secondary structure o bacteria, archaea and euk highly conserved structure tRNA structure-anticodon, amino acid attachment, cloverleaf that then forms 3D structure  ssRNA of 73-93 nt in bacteria and eukaryotes  cloverleaf structure in 2D  Twisted L shape in 3D  Most have G at 5’ end o All have CAA at 3’ end (aa 3’ prime)  Modified bases  TYC arm  D arm o Contains two/three D residues at diff positions  Extra arm o Variables in size, not present in all tRNAs  Anticodon arm o Wobble position  Slide 24-25 Translation  Initiation o mRNA and aminoacylated tRNA bind to small ribosomal unit o large subunit then binds o 30S subunit binds to IF1 and IF3, then mRNA binds  IF3 keeps subunits apart  IF1 blocks A site o Initiating codon AUG on mRNA, guided to position by shine-dalgarno seq  Region in mRNA o fMet tRNA binds to peptidyl site along with initiating 5’AUG  IF2-GTP binds too o Large subunit combines with 30S forming initiation complex  IF2 hydrolyzes GTP to GDP, then all IFs leave o Slides 36-38  Elongation o Successive cycles of aminoacylated tRNA binding and peptide bond formation until ribosome reaches STOP o Aminoacyl tRNA binds to elongation factor Tu (Ef-Tu) that carries GTP o Complex binds to A site of the 70S complex o After GTP hydrolysis, complex leaves ribosome  Ef-Tu is recycles GDP/GTP with help from EF- Ts o Now two aa tRNAs positioned  A site and P site o fMet is transferred from P site to A site  done by 23S rRNA  growing peptide transferred to aa on tRNA of a site  uncharged (deacetylated) tRNA fMet  now in P site o translocation of ribosome is final step  moves toward 3’ end  energy fro GTP hydrolysis on EF-G  leaves A site open for new aminoacyl tRNA  uncharged tRNA moves from P to E site then leaves  growing peptide on tRNA in P site  EF-G recycles GDP to GTP, next aa enters A site  Termination o Translation stop when stop codon encountered  UAA, UAG, UGA in A site  Triggers release factors 1-3 o The mRNA and protein dissociates and ribosomal subunits are recycled o Protein folding o RF1 and RF2 act the same but bind diff stop codons o Hydrolyze terminal peptide tRNA bond o Release peptide and tRNA fro ribosome o Cause subunits of ribosome to dissociate so that initiation can begin again and IF3 binds the small subunit o Euk only have 1 release factor  Features o Large energy cost  1 ATP and 3 GTP per aa o can be rapid when accomplished on clusters of ribosomes called polysome o tightly coupled transcription  translation can begin before transcriptio


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