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Biochem 153B Final e

implications of base aromaticity on nitrogen basicity? purines: N1, N3 pyrimidines: N1, N3, N7, but not N9 because the electrons are delocalized due to aromaticity -- protonation would lead to loss of stability
implications of base aromaticity on base stacking? aromatic bases are planar and can stack efficiently p-orbital delocalization leads to adjacent stacked bases strongly attracting with induced dipole interactions (leads to 3D structures)
What are the relative stability of the tautomers? keto tautomer predominates over enol because O have greater electronegativity than N, so bond is more polar (OH > NH)
significance of tautomer stability to basepairing? If enol form pre-dominated, could get not-watson crick base pairs
free nucleotide tautomeric stability the standard free energy of enol tautomer is greater than keto and you want the lowest, so keto predominates for free nucleotides as well
significance of Rosalind Franklin’s data (xrd) and Bragg’s law (wavelength and distance) in formulating watson crick base pairing model glycosidic bonds were symmetrical about pseudodyad axis which allows for any order of base pairs without disrupting helix AT and GC are same width distance between C1' atoms is same for all base pairs
significance of Chargaff’s rules in formulating watson crick base pairing model chargaff's rules: %G = %C and %T=%A therefore, G pairs with C and T pairs with A
Why does the double helix only accommodate watson crick two basepairs? the Z glycosidic bonds are related by a pseudodyad axis and the distance between C1' atoms is the same, so AT and GC are same widths and won't disrupt the helix
How do the overall shapes of A- and B-form helices differ and how do the different sugar puckers contribute to this? A has less rise than B A is C3' endo and B is C2' endo A has large major groove and shallow minor groove base pairs are tilted in A DNA A has a hole in the middle
What is the role of water in stabilizing B- relative to A-DNA? B DNA is stabilzied by interaction with H2O and H2O binds more tightly to B DNA ==> dehydrating conditions lead B --> A has 2 layers of H2O in the major groove -- bridges adjacent base pairs on opposite sides and bridges layer 1 of water molecules
What is the role of the 2’OH group in stabilizing A- relative to B-RNA? RNA has steric crowding in C2' endo conformation due to 2'OH group, so the sugar pucker is forced into B form to minimize this
How is the Watson-Crick model a hybrid between A- and B-DNA?
Why are Z-form helices restricted to sequences that alternate between purine and pyrimidine residues and at high salt concentrations? lessens interphosphate electrostatic repulsions
Z structure purines: C3' endo, syn pyrimidines: C2; endo, anti
B structure purines and pyrimidines are the same C2' endo, anti
A structure purines and pyrimidines are the same C3' endo, anti
What are the physical parameters that favor single-stranded DNA? greater electrostatic repulsion and entropically favored
Conformational entropy - what is it and what suggests that it is the main entropic contribution to basepair formation allowable dihedral angles for each bond -- more for dsDNA than ssDNA, so must be contributor for base-pair formation (dsDNA formation)
Translational entropy - what is it and why does it increase when DNA melts (ssDNA formation) potential positions in space: more for ssDNA than dsDNA because 2 strands instead of 1 limits spots
how does Electrostatic repulsion between the strands affect DNA melting more electrostatic repulsion means the phosphate backbone repels and will favor denaturation -- higher salt concentrations insulate DNA from the repulsions and favors dsDNA; low salt - higher electrostatic repulsions and favors ssDNA
What are the physical parameters that favor double-stranded DNA H-bonds decrease enthalpy and base stacking interactions
Hydrogen bonding between the strands - why is this a minor factor in aqueous solution for formation of dsDNA H bonds are a minor factor because in an aqueous environment can form equally enthalpically favorable H-bonds with water and to itself
Stacking - what is it and how does it stabilize double-stranded DNA induced dipole interactions between base pairs and it's enthalpically favorable for dsDNA also hydrophobic interactions which drives dsDNA entropically --> lipid aggregation releases water from ordered stat
What is the physical basis for the hyperchromic shift and what is it? absorbance of DNA solution increases during DNA denaturation as a result of disruption of electronic interactions between adjacent bases
Why does the shape of the melting curve indicate that melting is cooperative and what do we mean by cooperativity sigmoidal-shape indicates cooperativity: each step increases the favorability of the next step
what info can we get from a melting curve 1/2 max absorbance means equal [dsDNA] and [ssDNA]
how was marmur doty equation determined experimental observations that Tm = 0.411(%GC) + 69 = 41.1XGC + 69 can add salt correction term: 16.61log([Na+]/0.165)
When is it appropriate to use marmur doty with long and complicated duplexes (various base pairs)
under what circumstances does marmur doty break down? for small and/or noncomplex duplexes (repeat base pairs) due to length effects and nearest neighbor effects
What is the physical meaning of the values in the nearest neighbor table? shows how each base pair affects the adjacent base pairs
How do we use the values in the nearest neighbor table to calculate the free energy, enthalpy, and entropy of helix formation and to determine melting temperature plug the sum of each into Tm = H / (S + Rln(Ct/4)) where R = 0.002 kCal/molK and Ct = 5e-2
Why does the length dependence of melting temperature vanish for sufficiently large DNA? nearest neighbor effects and length effects diminish and cancel out and only account for small differences
How does DNA profiling work? PCR to amplify DNA and look at number of repeat sequences for each individual (varies from human to human)
Why does DNA profiling depend upon the ability to calculate DNA melting temperatures accurately? requires PCR -- hybridization in PCR only works accuratelt if just below Tm because if too high, duplex won't form and if too low, duplex will form slowly and nonspecifically.
How can topology help us to understand the properties of DNA? it influences the DNA structural transitions such as the formation of denatured bubbles (DNA melting) and nucleosome assembly
What is the physical meaning of Twist, T # helical turns = N*helical twist/360 *N = number base pairs
What is the physical meaning of writhe, W # of times ribbon's axis crosses itself left handed triple = +1 right handed triple = -1
What is the physical meaning of linking number, L L = T + W topologically invariant
How do interwound and toroidal supercoils contribute to the Writhe?
What do we mean when we say that Linking number is a topological invariant? it cannot change because the number of times the straws are linked cannot change without -- changing the connectivity
How do type I topoisomerases change linking number? breaks 1 strand and passes other strand through break and reseals; each cycle changes L by +/- 1
How do type II topoisomerases change linking number? breaks both strands and passes double stranded segment through and reseals; each cycle changes L by +/-2
How do topoisomerases preserve the free energy of the phosphoester bond in DNA? ALL proceed through covalent intermediates which involve ester linkages between phosphates at the break and tyrosines in the active site -- phosphotyrosine linkage preserves energy of phosphoester bond and hold broken end in place
How is writhe related to free energy? lowest free energy topological state is W-->0 -- increased free energy as a result if increased writhe
How can supercoiling drive DNA melting? if DNA is relaxed or positively supercoiled, a bubble increases |W| and is disfavored if DNA is negatively supercoiled, bubble formation will decrease |W| and is favored
How is nucleosome assembly influenced by DNA topology? histones increase superhelical strain (W) by 2 in regions not near nucleosomes until topo I or topo II releases it, but overall W decreases by 2
What do the Cairn’s experiments tell us about the nature of DNA replication in E. coli? 1. e. coli genome is 1 huge circular chromosome and replication occurs semi-conservatively from a single origin on each chromosome 2. replication occurs bi-directionally
What three properties are common to all DNA polymerases? 1. catalyze polymerization of deoxyribonucleotides in 5'-3' 2. requires a template 3. requires a primer
What is the basic chemistry of the DNA polymerization reaction? DeltaG is slightly negative, with a high K value, therefore if [PPi] > 200, than dNTP would catalyze net pyrophosphates degradation --> maintain low PPi levels in-vivo
Given that DNA polymerases use 5’ NTPs as substrates, why does it make sense to always polymerize DNA in the 5’ to 3’ direction? nucleotides are added in 5'-3' because the 3' OH of synthesized DNA can perform a nucleophilic attack on an incoming nucleotide because the beta and gamma phosphates are a good leaving group
Why is it useful for the end of the primer/template duplex in the DNA polymerase active sites to assume an A conformation? It sets up the active site to have a particular diameter to allow H-bond contacts (stabilize and position the duplex) for polymerization to occur -- these contacts are only able to be made in the more narrow minor groove of A-DNA
How do metal ions catalyze the polymerization reaction? 2 metal ion mechanism -- metal ions are used to catalyze phosphoanhydride bond by creating a near-optimal transition state via lowered entropy and results in a suboptimal product to stabilize the 3'-OH to form new phosphoester group and translocate
when do Mg ions bind during polymerization bind subtrate pre-initiation complex of reaction, during transition state, and products after reaction is complete
What is that makes the high fidelity of DNA replication so surprising? some non-watsoncrick base pairs are only about 100-fold less stable than watson-crick which suggests error rate should be 1 in 100
How do cyclic conformational changes in Pol I help it to achieve high fidelity? Klentaq clamps tightly pre-catalsis so wobble doesn't fit -- samples other base pairs, but only the wobble base pairs allow it to fully clamp
How does the 3’ to 5’ exonuclease activity of Pol I help it to achieve high fidelity? occurs close to site and allows it to erase mistakes and try again; nucleophilic attack by water on phosphate which frees the 3' hydroxy group so it can be correctly polymerized-- occurs in a different active site from polymerization
How does mismatch repair work to clean up remaining errors? after replication, DNA is hemi-methylated (parental is, new is not) which indicates which strand may need reparation
What is the problem and solution to the directionality problems of DNA Polymerase pol operates 5'-> 3', so how can DNA repair both strands? soln: semidiscontinous DNA synthesis: leading strand is polymerized smoothly and lagging goes 5'-3' in opposite direction in segments, so bubble can still move in the same direction
What is the priming problem and solution for DNA polymerase? all DNAP requires a primer, but where does this come from? soln: use RNA primers
How did Okazaki prove that Okazaki fragments are primed with RNA? with alpha32P labeled dNTPS and isolated fragments and exposed them to alkane hydrolysis which showed a ribonucleotide with 3' 32P (indicated RNA/DNA junction)
Why might it be useful to prime Okazaki fragments with RNA? DNAP needs primer so it can be used, but ultimately must be able to replace/degrade it leading strand only need this 1x, okazaki need for each fragment
What are the major components of the E. coli replisome and how do they work together to bring about semidiscontinuous DNA synthesis at a replication fork? helicases, ssDNA binding proteins, primosome, DNAPIII, topoisomerase, DNAPI, DNA ligase
helicase role in E. Coli replisome unwinds DNA at rep fork in reaction couple to ATP hydrolysis
ssDNA binding proteins role in E. Coli replisome binds and stabilizes DNA in ss conformation after helicase melts it to prevent self pairing
primosome role in E. Coli replisome deposits RNA primer ever 1k-2k base pairs behind moving replication fork --> needs 3'-OH so RNA primers synthesize for keading
DNAPIII role in E. Coli replisome the replicase --> main polymerase responsible for DNA polymerization in e. coli polymerizes 5'-3' and edits 3'-5'
topoisomerase role in E. Coli replisome relaxes positive supercoiled DNA ahead of replication fork (topo I or II), topo II decatenates final product
DNAPI role in E. Coli replisome replaces RNA primers with DNA by nick translation -- fills in RNA primer with dNTPs
DNA ligase role in E. Coli replisome joins okazaki fragments -- catalyzed ligation between last dNTPs (3'-5' of dNTPs)
How do hexameric helicases (e.g. DNAB helicase) achieve strand separation? 6 copies of a single polypeptide arranged in a spiral staircase with room in the middle for ssA-DNA where each subunit makes H bods to phosphates n 2 bp achieves strand separation by a hand-over-hand mechanism
3 steps of hand over hand mechanism in DnaB helicase (a hexameric helicase) 1. hydrolyzes ATP and releases ADP which triggers the release of top subunit from adjacent 2. released subunit remains anchored by NTD 3. flexible linker between NTD and CTD allows released subunit to move down and clamp bottom subunit with ATP
Why does the primosome consume ATP even when it is not synthesizing primers?
What are the major modules within Pol III and what do they do? core: alpha, epsilon, sigma sliding clamp: beta2 clamp loader: gama2, delta. delta'. psi, rho2
role of core modules in DNAPIII alpha = catalytic epsilon = 3'-5' editing exonuclease sigma = structural role
role of sliding clamp (B2) in DNAPollII increases processivity by dimerizing and forming clamp around DNA strand so it can accept B and A DNA
role of clamp loader in DNAPIII puts clamp on and links leading and lagging core polymerases
How are processivity and efficient recycling of Pol III at odds with one another? processivity requires high binding affinity of the clamp, but efficient recycling requires ability to come off and reuse -- must regulate clamp and core affinity
How do the clamp and the clamp loader work together to ensure both processivity and efficient recycling? 1. ATP opens and allows clamp to bind 2. clamp binding primer stimulates ATP hydrolysis, release of ADP, release of clamp loader and closure of clamp 3. DNA synthesis can occur 4. when core see okazaki, loses affinity 5. primosome syn new primer
What are the roles of DNA polymerase I and DNA ligase in finishing up the process of DNA replication? DNAPI excises RNA primers by nick translation and DNA ligase seals nick to complete sequence
Why are topoisomerases necessary for DNA replication? relax positive supercoils ahead of replication fork and decatenates final product
Why do we specifically require topoisomerases that can reduce the linking number and why do we require type II topoisomerases? as strands separate, T gets lower and lower, so without topo, W would get larger and eventually movement would be prevented topoII decatenates daughter chromosomes
How do OriC and DnaA work together to initiate DNA replication? DnaA recognizes and binds oriC and melts DNA to form replication bubble and then DnaB and DnaC can unwind to form pre-priming complex
In what ways do the initiation and elongation phases in eukaryotic replication resemble those of bacterial replication and in what ways are they different?
Why do eukaryotic chromosomes require multiple origins of replication Eukaryotes have multiple origins of replication because the genomes of eukaryotes are larger than prokaryotes and their DNA polymerase is slower
How does telomerase enable eukaryotic cells to solve the end replication problem? RNAH cleaves RNA duplex and poldelta and DNA ligase fills in an you end up with a fragment of DNA thats missing so telomerase serves as a template the RNA primer can be synthesized and the poldelta can resyn the missing (otherwise cells die)
What is the significance of telomerase in aging and disease? mutations in telomerase lead to premature telomere shortening (which means losing information) and ultimately leads to cell death
How do UV radiation lead to DNA damage? causes covalent bonds in cyclobutyl ring to be shorter than normally stacked bases -- distorts DNA
How does alkylation lead to DNA damage? any nucleophilic atom on DNA can be alkylated, sometimes leading to changes in base pair specificity
How does oxidation/hydrolysis lead to DNA damage? deamination -- replaces NH2 with O oxidative: forms reactive oxygen species which can cause structural mutations or directly attack and break the DNA backbone
Why is it important for DNA to contain thymine instead of uracil? sometimes spontaneous C --> U deamination, and if U is supposed to be there, no way to check if it came from daminantion or if it's intentional
How does DNA damage affect the templating properties of the bases? can create new H-bond donors and acceptors, so will base pair differently than watson-crick base pair
Direct reversal - how does AGT reverse alkylation and how does it serve as a sensor of the overall alkylation state of the genome? can remove methyl and ethyl by transfer; not an enzyme because gets irreversibly modified by reaction --High levels of alkylation, alkylated AGT, act as a transcription factor to activate the gene that encodes the DNA repair protein
what is AGT O6-alkylguanine-DNA-alkyltransferase
What is the overall strategy of base excision repair? nick translation: in bacteria, the exonuclease and polymerase functions are both provided by polI through the process of nick translation -- size of gap can be 1-a few nucleotides
How do DNA glycosylases recognize their cognate damaged bases? damaged base has a structural mutation causing it to flip out of normal helix, exposing it for recognition
What properties are common to all RNA polymerases? 1. synthesize RNA is 5'-3' direction 2. require a DNA template 3. can synthesize RNA strands de novo 4. exhibit processivity
What is the difference between the bacterial core polymerase and the holoenzyme? core can only initiate transcription randomly (from nicks/gaps), need holoenzyme for regular initiation
components of RNAP core α2, β, β', ω
components of RNAP holoenzyme α2, β, β', ω + σ
role of α2 in RNAP core anchors β, β' together
role of β, β' in RNAP core active site forms at interface of the subunits
role of ω in RNAP core inessential, but may be a chaperone for folding of RNAP
role of σ in RNAP holoenzyme RNAP binds to DNA randomly and σ recruits RNAP to promoter region
How was DNase I footprinting used to identify the lacUV5 promoter? missing bands on gel showed where DNA did not get fragmented and showed where RNAP was bound to DNA
What are the basic elements of a bacterial promoter and how were they identified? operon, -35 region, TATA (pribnow)/-10 region, initiation site
How are different factors used to direct core polymerase to different genes? help bacteria cope with different types of stress
What are the 6 steps in the transcription cycle? 1. closed complex formation 2. open complex 3. abortive initiation 4. promoter clearance 5. elongation 6. termination
What are the characteristics of the closed complex formation in the transcription cycle? kinetically unstable
what are the characteristic of the open complex in the transcription cycle kinetically stable -- formation is essentially irreversible RNAP is functioning like a helicase (sig 54 requires ATP but sig 70 does not)
what are the characteristics of abortive initiation in the transcription cycle? bubble moves to the right as transcript is made can make 100s of transcripts before proceeding to next step RNAP maintains contact with the promoter
what are the characteristics of promoter clearance in the transcription cycle? occurs when transcript achieves ~10 bp ejection of sigma increases stability so enzyme is highly processive and releases RNAP from promoter
characteristics of elongation in transcription cycle?
characteristics of termination in transcription cycle? signalled by GC rich inverted repeat because RNAP transcribes through palindrome and then pauses on formation of AU rich heteroduplex and there's no room for hairpin in the active site so transcript is released
How does DMS footprinting work and how was it used to demonstrate contact between RNA polymerase and the major groove and open complex formation? methylates N7 or N3 to destabilize glycosidic bond -- methylated gets cleaved see disappearance of bands on either side of pribnow box without RNAP and can see protection of G residue corresponding to -35 region; add RNAP and holoenzyme protects the G
What are the the primary and secondary channels and how are they involved in transcription? Primary: is the opening where the DNA goes through Secondary: where incoming NTPs go and have easy access to Mg ion
How is bubble size controlled by a “zip lock”? beta and beta' act as zippers and moves the bubble with RNAP with metal ions
what does the pre-translocation state of RNAP look like? The Mg2+ ion is next to the last phosphodiester bond in the RNA
What is the role of metal ions in transcriptional elongation? coordinate with amino acids and can deprotonate and create optimal bond geometry so the phosphoester bond is easier to form
How does σ mediate the binding of core RNAP to the promoter? σ domain induces DNA from -11 to +2 to melt and allow template strand in ssDNA region to enter active site and stay near metal catalyst
How does competition for the space under the  flap lead to abortive initiation and control the transition the transition from the ITC to the TEC? the σ 3-4 linke rregion blocks further initiation of transcript and leads to abortive transcripts -- once transcript can push linker out of the way, the σ factor is released and there is promoter clearance
what is the purpose of abortive initiation provide a step by which to regulate transcription and and σ3-4 linker in active site acts as an extra binding interface for the 1st nucleotide so RNAp does not need a primer
How does microcin J25 inhibit RNA polymerase? and what does this tell us about transcription blocks secondary channel -- tells us where NTPs enter
How does rifampicin J25 inhibit RNA polymerase? and what does this tell us about transcription binds inside bubble and creates a steric clash after 2-3 residues
What is the role of Lac operon negative regulation? transcription is decreased by the presence of the repressor and bad -10 region
What is the role of lac operon positive regulation? 1. presence of lactose inactivates repressor 2. absence of glucose activates CRP which is an activator
What is it about the lac promoter that makes it a bad promoter? Why is a bad promoter good? It is different than the consensus sequence. It allows ensures the lac promoter is inactive in the absence of cAMP-CRP (Glucose). Positive regulation.
Why is the major groove a richer source of sequence information than the minor groove? has more space between the backbones, so the bases are more exposed and more sequence information is available
How do HTH motifs recognize and bind specific DNA elements? one helix acts as a molecular outrigger to stabilize the recognition sequence and the other is the 'recognition helix' which interacts with the DNA
What is AR1 and how was it defined? activation region 1 - has mutations which prevent CRP from activating transcription without interfering with DNA binding (positive control mutations)
What is the recruitment model? (RNAP) 1. CRP recruits RNAP to promoter 2. CRP contacts RNAP alpha-CTD when both are bound to lac promoter 3. CRP activates lac promoter via interaction with RNAP CTD
what does 'recruitment' mean in RNAP? energetically favorable interaction between AR1 of CRP and CTD alpha subunit lends stability to RNAP and promoter
what is alpha CTD required ofr? not required for transcription, but is required for CRP activation
How was it determined that the AR1 domain of CRP interacts with RNA polymerase? Label transfer experiment - they labeled the CRP and transferred it to RNA polymerase (mapped to alpha subunit of CTD)
what experiment showed that RNA polymerase interacts with CRP? Reverse experiential: Igarashi experiment: they truncated the C-terminal domain of the alpha subunit
How were the eukaryotic RNA polymerases discovered? fractionation of frog nuclear extract with anion exchange chromatography and compared RNAP activity with and without α-amanitin (an RNAP inhibitor)
What are the three main eukaryotic RNA polymerases and what genes do they transcribe? RNAPI: class 1 genes RNAPII: class II genes RNAPIII: clas III genes
what does eukaryotic RNAPI do? ~50% of cell's transcriptional activity and encodes 45s rRNA precursor class I genes
what does eukaryotic RNAPII do? ~10% of transcriptionala ctivity transcribes genes encoding mRNAs and small RNAs subject to explicit regulation class II genes
what does eukaryotic RNAPIII do? accounts for 40% of cells transcriptional activity transcribes genes encoding tRNAs, 5s rRNAs, and small RNAs promoter is often internal to transcribed region does class III genes
What is the relationship between bacterial core polymerase and the eukaryotic polymerases? eukaryotic polymerases contain homologues of all bacterial core subunits --> structures f catalytic sites and mechanisms of catalysis are highly conserved. but eukaryotes contain additional subunits
What do class II core promoters look like? contain TSS TATA/pribnow box at -30 INR (initator element) at +1 Downstream promoter element (DPE) at +3 usually have 2-3 of elements ++ CRM and exons
what is the CRM in class II basal transcriptional machinery binding site for regulatory factors and can be upstream or downstream from core promoter elements
What is the class II pre-initiation complex and how is it assembled? = bacterial closed complex contains TFIID, TFIIB, TFIIA TFIIF, TFIIE, TFIIH, Poll II and CTD
How does TFIID recognize the TATA box? has ~15 subunits including a TATA binding protein (TBP) TBP binds minor groove which must open to accomodate a 10-strand anti-parallel beta sheet
describe the TATA binding protein (TBP) of class II eukaryotic transcriptional machiner is saddle shaped with helix as the 'horse' induces a sharp bend to recognize the sequence with a Phe in the stirrups and wedges the base pairs apart to create the bend
What is the role of the TFIIH helicase and kinase activities in promoter opening and promoter clearance? as a kinase: CTD phosphorylation is linked to eukaryotic promoter clearance as a helicase: open complex formation at class II promoters requires TFIIH as a helicase
What do we mean when we say that sequence transcription factors are modular in nature? 2 independently folding globular domains connected by a flexible linker (an activation/repression domain and a DNA binding domain)
How do various types of DNA binding domains present recognition helices to the major groove? 1. Leucine zippers (bZip domain) are the pseudoheptad repeat which makes side contacts all down the alpha helix 2. Zinc finger (cys2his2ZF) has 2 antiparallel beta strands and an alpha helix which bind tetrahedrally to a Zn ion to stablize
What are three basic ways in which coregulators modulate rates of transcription? 1. assist in basal machinery recruitment (e.g. the mediator) 2. modulate regulation by RNAP 3. via chromatin structure
What is the molecular nature of the Mediator? has a tail, middle, CDK8 region, and head region - each with different functions
where do dominant suppressors bind the mediator and what does this mean? binds the head region which interacts withRNAP to help find the promoter DNA region contributes to gain of function mutations which means increased mediator function
where do recessive suppressors bind mediator and what does this mean? bind the CDk8 region which is antagonistic to contacts and contributes to loss of function mutations
What is chromatin? complex of DNA, histones, and non-histone proteins ound in the nucleus of eukaryotic cells
what are the 2 types of chromatin heterochromatin and euchromatin
what is the significance of heterochromatin condensed chromatin -- region of chromosome which is transcriptionally inactive during interphase (stains dark and dense)
what is the significance of euchromatin 'normal,' not condensed chromatin --> transcriptionally active during interphase and stains diffusely
what is the higher orders of DNA packaging chromosomes ar emade of condensed chromatin wrapped on nucleosomes
What is the structure of the nucleosome? bead like structure on eukaryotic chromatin --> short length of DNA wrapped around core histone proteins
the function of the mediator is closest to which components of the bacterial transcriptional machiner? the CTD of core polymerases alpha subunit
How are contacts between the DNA and histones maintained? the 8 histone proteins: 2 H2A-H2B dimers (a histone fold pair) 1 H3-H4 tetramer
What is the histone code hypothesis? pattern of post translational modifications on histones that can influence gene function (activation or repression)
How do post translational modification result in gene activation and repression? acetylation of lysine usually means activation methylation of lysine can activate or repress phosphorylation of serine regulates chromosome condensation and usually results in gene repression
How do bromodomains facilitate transcription? recognize acetylated lysines on histones --> protein sits on histone and LysAc interacts in the binding pocket near the bromodomain which forms H bonds recognizes acetylated histones bc if no acetylation ==> no transcription
How do we know that acetylation of histones causes gene activation? Gcn5, which is required for RNAPII is a HAT ==> Evidence that transcriptional machinery can acetylate histones
How is DNA methylation passed from one generation to another? methylation of cytosine results in gene silencing DNMT -- de novo or template and can be passed down via memory of methylation state
What is meant by the term ‘epigenetic’?
What do the locations of introns tell us about the origin of introns (2 theories)? 1. Exon shuffling: It is a process through which two or more exons​ from different genes can be brought together ectopically, or the same exon​ can be duplicated, to create a new exon​-intron structure 2. Propagation of junk DNA - transposons
How do conformational changes in the spliceosome serve to bring the splice sites together and what is the role of ATP in these conformational changes? forms a lariat and then cuts lariat out (2 sequential transesterification rxns) ATP ensures specificity despite energetically neutral reactions (conformational changes of the spliceosome complex)
What three elements of the pre-mRNA transcript are required for the splicing reaction? 5' splice site, branchpoint, and 3' splice site
What does the branch point nucleotide contribute to the transesterification reaction? 2' OH group (nucleophile)
What does the ara-A experiment show us about how the branch point works? the C' epimer preferentially selected for the bulge loop. hydroxyl was not in right position (stereochemistry is wrong), so reaction does not occur
How does a metal specificity switch show a catalytic role for metal in splicing? subbed hard base with a soft base and did not get final spliced complex when removed metal ions (EDTA) no complex formation or splicing
Within the spliceosome, what is the purpose of the U6 ISL? holds the Mg for splicing rxn
What is the structure and purpose of the 5’ cap? structure: 7mGppp (7 methyl guanosine triphosphate) purpose: 1. makes 5' end resistant against 5’ to 3’ exonucleolytic cleavage 2. helps ribosome recognize mature mRNA
How is capping coupled to transcription and what is the advantage of this coupling? ensures efficient channelling of transcript from transcriptional machinery ti capping machinery (phosphorylated CTD is a docking site for capping enzymes)
What series of events leads to the formation of the mRNA 3’ end? cleavage and polyadenylation specificity factor (CPSF) cleavage stimulatory factor (CStF) cleavage factors (CFs) PAP (polyA polymerase)
What is the purpose of the polyA tail? protect 3' end of mRNA fron exonucleases directs transport of mRNA from nucleus to cytoplasm assists in recognition of mRNA by ribosome
What advantage does alternative splicing have over transcriptional regulation as a way of regulation gene expression? can regulate splicing as opposed to transcription -- allows for the same mRNA to encode different proteins with varying or even opposite functions
How is splicing regulated in Drosophila sex determination? females have sxl gene which allows for complete TRA protein which can then allows the recruitment of splicing factors that recognize the non-consensus sequence branch point of the dsx gene (get exons 1234) males do not have complete TRA (exons 12356)
What what is the role of guanosine in group I self splicing RNA? guanosine acts as nucleophile which attacks 5' splice junction
Why do we think that the spliceosome is probably a remnant of the RNA world? shows proteins were not needed and solves problem of how proteins and nucleic acids which encode proteins could evolve simultaneously
How was RNA interference discovered? trying to make darker red petunias by adding an extra copy of the gene but ended with white
How did Fire and Mello demonstrate the nature of the trigger? injecting sense and anti-sense and mixed RNAs into animals saw that sense and antisense resulted in null phenotype but no null phenotype when using introns
What are the differences between the siRNA and miRNA pathways? The miRNA does not have complete complementarity to the target mRNA -- siRNA has complementarity, but uses RISC to cleave to suppress transcription
How is the genetic code designed to minimize deleterious effects of mutations? degeneracy and non-random ordering of base pairs
wobble hypothesis and why having a wobble base pair on a tRNA would be advantageous A- site imposes w/c base pair geometry on first 2 base pairs but is more lenient with the third -- makes it possible to recognize 61 sense codes with only 31 tRNA species
How might tRNA covalent modifications contribute to tRNA function? have pseudouridine and dihydrouridine, both of which are dispensable -- unclear what purposes are
What are the four branches of the tRNA cloverleaf? D-arm, T,pseudouridine,C arm, variable arm, and anti-codon arm
How does the cloverleaf fold up to form the L-shaped tRNA? 2 A-form helices in an L-shape A-form due to the 2' OH group
What is the purpose of tRNA charging? 1. provides amino acid with necessary adaptor 2. activates aminoacids carboxyl group for peptide bond formation
what is tRNA charging attachment of amino acids to cognate tRNA, catalyzed by aminoacyl-tRNA synthetases
What are the two steps in tRNA charging? 1. formation of aminoacyl-AMP 2. conversion of aminoacyl-AMP to aminacyl-tRNA
How do aminoacyl-tRNA synthetases recognize their cognate tRNAs? class I: read anticodon sequence directly class II: recognize features in acceptor stem instead of anticodon
Why is editing required to ensure high fidelity tRNA charging and how does this editing work? editing is required because amino-acyl tRNA synthetases are responsible for reading the genetic code editing works by sieving out too large and adenylation/hydrolysis of too small
What does the fragment reaction tell us about the ribosome? suggests ribosome is a ribozyme
How was the Yarus inhibitor used to prove that the ribosome is a ribozyme? yarus was a ts-analogue so used for xray crystallography to show that nothing else is nearby to catalyze reaction
What evidence suggests that the ribosome peptidyl transferase works by positioning the substrates and not by chemical catalysis? rate enhancement occured without change in activation enthalpy -- showed change occured via entropy increase as opposed to enthalpy decrease
How is the S/D sequence used to place the start codon in the P-site? via formation of duplex between s/d and 3' end of 16s rRNA to position start codon in P-site
Why are polycistronic messages common in bacteria, but not eukaryotes? shine dalgarno sequence will guide ribosome to desired gene in each message, but eukaryotes just scan for the first start codon (AUG)
How does a GTPase drive initiator aa-tRNA delivery? gtp-ases couple conformational changes to GTP hydrolysis and act as a motor to drive process of protein synthesis forwards
Chain elongation - How do GTPases drive aa-tRNA delivery and translocation? with EF-Tu to efficiently place aminoacyl-tRNA into A site and drive translocation of EF-G
Chain termination - How do release factors trigger polypeptide release?
How does a difference between bacterial and eukaryotic IF-2 aid regulation? eukaryotic eIF-2 can be phosphorylated to form a dead end complex with eIF-2B and will be sequestered out of the cycle
How does eIF-2 phosphorylation control globin synthesis and mediate the response to amino acid starvation? HRI is inactivated by heme -- without heme can autophosphorylate eIF2 and sequester it out of cycle GCN5 is activated by amino acid starvation and prevents inaccurate synthesis by sequestering out the eIF2 and preventing translation
What is the fundamental difference between the logic of the de novo purine and pyrimidine pathways? purine starts with PRPP and builds purine onto the sugar while pyrimidines build the free pyrimidine ring and add PRPP later
Why do we have both salvage and de novo pathways? salvage: more favorable to reuse bases than synthesize new ones and if the bases are not salvaged, they must be broken down de novo: if not enough bases are not available through degraded nucleic acids or the diet have to be able to make them anyways
why is the purine salvage pathway so important purine catabolism products are toxic to the cell
Where does PRPP come from and what is the reason for using it in all nucleotide biosynthesis? comes from activated form of ribose via the pentose phosphate pathway uses 2 ATP
Pyrimidine biosynthesis De novo is a major pathway Base is synthesized, then attached to ribose UMP, a typical nucleic acid, is converted into other pyrimidines
Purine biosynthesis Salvage is a major pathway Base synthesized while attached to ribose IMP is common intermediate for AMP and GMP, but itself is not a typical nucleotide
Determination of the pathway - How was pigeon poop used to determine the metabolic sources of the purine ring atoms? fed pigeons a 13C labeled compound and purified uric acid and fragments to see where label was using mass spec
Why are amino groups generally harvested from Gln rather than obtained from free ammonia? ammonia uses glutamine binding to channel for increased metabolic efficiency and avoids need to accumulate labile/toxic intermediates
What is the Ntn domain? N-terminal nucleophile -- glnase domain
Why is it important to start out with the alpha anomer of ribose in the Amidophosphoribosyl transferase reaction? because the reaction that converts PRPP from alpha into beta-5 phosphoribosylamine is an SN2 reaction, which inverts stereochemistry about the nucleophile -- so must start with the opposite of what we need
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