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59 Cards in this Set
- Front
- Back
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what are the differences bw DNA rep and RNA tc
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- no primer needed (for tc)
- uracil - continuous vs. discontinuous - ribose vs. deoxyribose - different enzymes (RNA vs. DNA Polymerase) - creates an RNA strand instead of a DNA strand - selective (tc is selective) - only uses one strand as a template (tc) - less accurate (no proofreading) - no permanent base pairing -tl=dNTP, tc=NTP |
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GENERL main dif. in tl and tc bw proks and euks
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in prokaryotes, transcription and translation can happen simultaneously in the same space
in eukaryotes, tc and tl are sep'd by time and space |
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what is the coding strand and the template strand? which one is the mRNA complementary to and the same to?
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the coding strand is what the mRNA seq would be if the T's were replaced with U's
the template strand is complementary and antiparallel to the coding strand |
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introns:
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are NON coding sections of DNA
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what direction is the template strand read and which direction is the mRNA made in?
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mRNA made in 5'->3' direction
template read from 3'->5' |
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role of RNAp?
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separate DNA strands and syn RNA (doesn't need an external helicase!)
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bac RNAp vs euk RNA?
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bac = 5subunits in the RNAp
euk = 3 separate dif RNAp's RNAp II= mRNA and snRNA |
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on what side of the RNAp is the DNA under/overwound (which is upstream and downstream)?
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upstream (where DNA feeds in) = underwound
downstream (where DNA feeds out) = overwound |
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what are the four (prokaryote) elements a protein encoding gene can be divided into?
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1) transcriptional control sequences
2) promoter 3) transcript 4) termination sequences (protein coding frame = open reading frame) |
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what are the to upstream regions? what are these sites?
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-35 = TTGACA
-10 = TATAAT +1 = TC Begins these are binding sites that allow TC to be initiated |
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apoenzyme and holoenzyme:
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the apoenzyme has no specificity, so it needs the sigma factor( a protein)
apoenzyme + sigma factor = holoenzyme |
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describe the initiation process of TC
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The RNAp binds to the closed promoter complex. (sigma factor binds the apoenzyme=haloenzyme --> binds the -35 and -10 regions)
It opens the DNA (1-1.5 helical turns or 12-13bp) to allow one strand to act as a template 2 NTP's join together to act as a mini primer |
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how is the open form more favourable?
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complex becomes open bc it is thermodynamically favourable: micro environment aa's bind to single stranded DNA better enzyme has high affinity for promoter especially in single stranded form |
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how does the sigma factor fall off?
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as tc begins, the promoter region leaves which disrupts the affinity of the sigma factor (since it has high affinity for the promoter)
- the apoenzyme can bind anywhere but has short half-life (for bonding to DNA) the holoenzyme has long life which allows it to go from closed to open |
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describe half life of apo and holo enzyme
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- the apoenzyme can bind anywhere but has short half-life (for bonding to DNA)
the holoenzyme (w/ sigma factor) has long life which allows it to go from closed to open |
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what are the two mechanisms for termination in prok's for RNA tc?
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1) rho dependent (rho = a protien)
rho protein binds to a 'rut' sequence, and then uses ATP to catch up to the RNAp - once rho caught up to RNAp, it pulls tc out which = halt in tc 2) rho independent - involves palindromic GC rich region - complementary strand finds the seq. which followed by A rich region - these complementary regions form stem regions = part of the termination sequence which lock up the RNAp (BC GC BINDS TO EACH OTHER) - aka a lil loop is formed which allows the transcript to fall out (AU bp's = weak) |
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what does length of transcript depend on?
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in rho dependent termination, it depends on how fast rho moves (to catch up to RNAp) which in turns depends on how much ATP is available in cell.
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Can rho independent/dependent processes work co-operatively?
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yes. if one fails, the other can come into play
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if the tc continues to go longer than expected (one of the termination mechanisms fail), will the protein be altered?
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NO! bc these extra sequences are apart of the 3' noncoding region
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what are 3 modes of regulation of TC in proks?
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1)basal tc set by promoter sequences
- the closer the promoter seq. is to the consensus seq that RNAp binds to= stronger the affinity/binding = increase in tc rate - spacing bw the UP elements (-10 and -35) 2) tc factors 3) sigma switching - can switch sigma factors - dif sigma factors have dif affinities for the promoters - sigma70 stesses ecoli via heating - sigma50 = in higher levels if coli in N-limiting environ |
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what is the main way for tc controL?
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at the level of the promoter region
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what happens if there was a mutation that caused sigma factors to bind to the -10 sequence directly in absence of the RNAp?
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DECREASE rate of tc bc it will occupy the -10 site = competitive inhibition with RNAp for that spot
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Cordycepin inihibits RNA syn, how? must it be modified to exert its effect?
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it inhibits RNA sys bc it has no 3'-OH. without this the next nucleotide cannot bind! (so can get incorporated into a strand but then terminates it from there)
it must be modified. a triphosphate must be added |
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differences in RNA transcription bw euk's and proks
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1)euk = chromatin -> DNA wrapped around histones so mustd deal with this, in proks its bare
2)tc in nuc, tl in cytoplasm, (do not occur simultaneously) 3)RNA must be spliced --> once made, introns must be cut out be4 mRNA leaves nuc 4)activation/fine tuning of tc can occur over LONG distances = ENHANCERS 5)mRNA is polyadenylated on the 3' end (polyA tail) --> gives it a longer half life 6)3 dif RNAp's, I, II, & III 7)also has a 5' cap |
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what do activators and repressors do?
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cycle bw condensed chromatin (gene is off) and decondensed chromatin (gene on)
gene on = histone acetylases chromatin remodelling complexes gene off = histone deacetylates chromatin remodelling complexes |
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what is involved in chromatin remodelling?
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HAT's = Histone acetyl Transferase --> can remove DNA off the histone by acetylating the histone protein (gene off --> gene on)
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what is the CTD? what happens for it to be activated? what does it do?
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c-terminus domain of RNAp II
- needs to be phosphorylated - recruits/binds all the players involved in RNA processing (5' cap 3'polyA tail) |
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what is the start site for tc? (promoter element)
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the TATA box, at position -30 (TATAAA)
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what is the consensus for the initiation / Inr site? (promoter element)
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YYAN(A/T)YY
(occurs at the +1 site) |
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describe other promoter elements like the CAAT box, GC box, response elements, and enhancer/silencer elements
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CAAT box sim to TATA box, w/in 100bp's upstream - a basal element
GC box - similar to CAAT box response elements - small molecs regulating tc, like steroids binding to DNA seq. enhancer/silencer elements - act at great distances (1000s bps) upstream and downstream - associated with very strong promoters |
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what do generalized tc factors do?
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(they're prots)
req'd for all RNApII mediated tc - bind to basal elements - req'd for polymerase binding (do not turn gene on/off) |
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what do specific tc factors do?
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regulate on or off state of a genes
specific for a or set of genes |
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is the TATA box necessary?
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no, if its not there just need CAAT,GC box, or both
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describe initiation of RNAp II initation
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- need TC factors to bind the TATA box and sep the DNA strands
- 1) TF II D/A = 2) allows TFII B to bind = 3) RNAp recruited and TF II F (processivity factor) TFIIF allows RNAp to finish its job 4) Then TFIIH comes = a) phosphorylate C-terminal domain (CTD = imp for processing) b)helps from going closed -> open 5) helicase using ATP does separation |
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do all these TFII prots stay bound once RNApII leaves the promotor?
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no! the prots all fall off. if the prots remained on the promotor it would inhibit further tc = regulation
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what is the only prot that remains stuck to the RNApII
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TFIIF
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what are the 3-post transcriptional modifications on mRNA in euks?
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1) 5' cap
2) introns spliced out 3) 3' end cleaved and polyadenylated |
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describe the 5' cap on euk mRNA. how is it linked to the mRNA? usually orientation?
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it is a 7-methyl guanosine, linked thru a 5'-5' triphosphate
allows the mRNA to be matured anti |
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why is 5' cap added right away?
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so it can start entering the cytoplasm while the splicing and other processing still is happening
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what is the CBC?
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cap binding complex
its role is to bind the 5' cap to keep the transcript close to the complex (to allow splicing) |
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what signals the addition of the poly(A) tail?
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the poly adenylation signal = AAUAAA sequence signals an endonuclease to make a cut 10 nucleotides downstream
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how is poly a tail added
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1) polyadenylation signal signals an endonuclease 10bps downstream from AAUAAA to make a cut
2) polyadenylate polymerase adds many Adenines to 3'-OH end using ATP to prevent degradation of 3' endand increase half life - doesn't need a template, just needs the 3'-oh site as a primer |
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introns exons and splicing
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introns = intervening RNA seq's that not part of final mRNA seq
exons = parts of transcript that is part of final mRNA splicing = process of removing introns and joining up exons |
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what is differential / alternative splicing?
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same gene: introns can be an exon for one product, but the same gene the exon can be intron for another product
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what is found at the ends of an intron usuallY?
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5'-GU--------AG3'
all within intron (GU and AG cover the borders) |
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what are splicesomes made of?
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snurps = small nuclear ribonucleic proteins
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what is snRNA? how does it guide the splicesome?
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- small nuclear RNA -->
- 1st, must be complementary to the splice site to guide the transesterification properly - it is continuously made and broken down -->made by RNAp based on level of tc that in turns governs tc |
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how does the splice some work?
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uses ATP
- an internal A before the AG-3' site on intron = a nuc onto the phospho linkage on splice site - |
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what happens to the spliced intron?
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forms a lariat shape. 2'5' phosphodiester linkage which soon gets degraded
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Group 1 & 2 introns = self spicing
- catalyst = the RNA itself - for it to work the RNA must adopt very specific 3D shape |
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describe Group 1 intron splicing
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Free flowing guanosine = acts as nuc
- attacks P group @ 5' splice site now intron only attached to exon at the intron 3' site - the 3-OH of the 5' exon acts as a nuc and completes the rxn |
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whats the difference bw group 2 intron splicing & group 1?
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Differce: for GROUP 2:
the nucleophile in the first step (5' exon site) and formation of lariat structure 2'5'phosphodiester bond (in group1, free flowing 3'OH G does attack, in group 2, an intron specific 2'OH A does it) |
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describe group 2 intron self splicing
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an intron specific adenine 2'OH group does a nuc attack on 5' splice site of intron = a LARIAT struc formed
- now a loop intron attached to 3' exon - 3'OH of the 5' exon acts as nuc attacks 3'OH completing the rxn |
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how many phosphodiester bonds does the initial adenine in group 2 self splicing have?
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at the end it has 3
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what are ribozymes?
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RNA molecs that catalyze chemical rxns
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what are examples of ribozymes?
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group 1 & 2 introns
RNase P (trna syn) rRNA = active site in peptidyl transferase in ribosome in part of the SrRNA large subunit |
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the ultimate gene product is not found on the coding DNA EVEN after processing. T or F
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True! actual final sequence of nucleotides not found on DNA.
you can have subsitution editing (change base) or insertion deletion editing |
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give an example of some subsitutino editing
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long/short forms of apolipoprotein B
CAA to UAA give different forms the intestine has an enzyme to cause this sequence change iT DEPENDS ON CELL LINE |
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RNAi...
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what!?
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