第八篇遗传信息的贮存及表达英文dna transcription

1、* Central Dogma- “One center, two basic points”* Basic features* Common to DNA replication* 1) Template, Unwinding and Torsion-relieving are necessary; * 2) Proceed only in the 53direction;* Cordycepin can prove this* Uncommon to DNA replication* 1) No need for primers* 2) NTPs instead of dNTPs; UTP

2、 instead of dTTP* 3) Lacking proof-reading activity ( error rate is 1 in 104 or 105 nts added )* 4) Specific regions (not all DNA sequence) can be transcribed* 5) To a specific gene, only one strand can be transcribed* Remember some nomenclature conventionsCentral DogmaTranscriptionTranslationReplic

3、ationReplicationRetro-transcriptionGene expressionCoding strand, Sense strand, Crick strandTemplate strand, antisense strand, Watson strandTranscriptionTranslation* The first nucleic acid synthesizing enzyme (polynucleotide phosphorylase, PNP )* In 1955, Marianne Grunberg-Manago and Severo Ochoa rep

4、orted the isolation of an enzyme that catalyzed the synthesis of RNA. For this work, Ochoa shared the 1959 Nobel Prize in Medicine with Arthur Kornberg* The real E. coli RNA Polymerase * In 1960, the true enzyme was identified by 4 separate groups: Sam Weiss at the University of Chicago, Jerard Hurw

5、itz, A. Stevens and J. Bonner. This enzyme required a template, used all four rNTPs as substrates and synthesized a product with a composition similar to that of the template, and it required Mg2+.* Common features* -DNA template: one strand is copied* -Substrate NTPs (GTP, CTP, UTP, ATP)* -Divalent

6、 cation (Mg2+ )* Differences between DNAP and RNAP Differences between DNAP and RNAP1) RNAPs can initiate synthesis which involves promoter recognition.2) RNAPs can melt the DNA duplex.3) RNAPs initiation is primed by a single nucleotide, not an oligo as is the case for DNAPs.4) RNAPs make multiple

7、contacts with the 2-OH of the incoming NTP.5) DNA scrunching occurs for RNAPs allowing abortive cycling while still retaining contact with the promoter.6) For RNAPs, the transcript is peeled away from the template; not so for DNAPs where the open cleft allows the duplex to extend out of the enzyme.7

8、) Initiation of synthesis is regulated by many proteins for RNAPs, but not for DNAPs.8) RNAP has no proofreading activity (error rate is 1 in 104 or 105 nts added)9) RNAP incorporates NTPs instead of dNTPs0) RNAP incorporates UTP instead of dTTPC All three classes of RNAs are transcribed by the same

9、 RNA polymeraseC In E.coli, RNAP is 465 kD complex, with 2 , 1 , 1 , 1 , 1 subunit C HoloenzymeC Core enzyme is 2 , 1 , 1 , 1 C InhibitorsC Rifampicin & Streptolydigina aa2a2 a2b a2b a2bb a2bb = core enzyme = core enzymea1bba2CORE ENZYMESequence-independent,nonspecific transcriptioninitiation+ve

10、getative(principal s) s70s70heat shock(for emergencies) s32s32nitrogen starvation(for emergencies) s60s60 SUBUNITinterchangeable,promoter recognitionThe assembly pathway of the core enzyme(the w subunit makes this more efficient)a1bba2s70s70RNAP HOLOENZYME -s70 Promoter-specific transcription initia

11、tionIn the Holoenzyme: binds DNA binds NTPs and together make up the active site subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. s recognizes promoter sequences on DNAThe sigma subunit CThe sigma subunit does two things:C (1) It r

12、educes the affinity of the enzyme C for non-specific DNA. C (2) It greatly increases the affinity of the C enzyme for promoters. E. coli also has six alternative sigma factors that are used in special circumstances Some of the sigma factors found in Bacillus subtilis Anti-sigma factorsC The importan

13、ce of anti-sigma factors has been established in recent years. These factors form complexes with their cognate sigma factor, thereby inhibiting its function. One example is FlgM, which is an anti-sigma factor for the flagellar sigma factor sF. Another example is Rsd, which is an anti-70 factor. It i

14、s not present in exponentially growing E. coli cells. However, when E. coli enters stationary phase, Rsd is synthesized and acts to block the activity of 70 thereby allowing S to associate with the core RNA polymerase and direct expression of stationary phase genes.C Control of sporulation in Bacill

15、us subtilis also involves anti-sigma factors and anti-anti-sigma factors!RNAP core structure from T. aquaticus.RNAP has a “crab claw” shape with a wide internal channel to bind DNA and RNA.RNAPs in Eukaryotes RNA polymerases I, II and III transcribe rRNA, mRNA and tRNA genes, respectively RNAP II In

16、hibitor Mushrooms of the genus Amanita make a toxic cyclic octapeptide called a amanitin (鹅膏蕈碱）lThis mushroom tastes good but eating it is deadly!l 6 to 24 hours after eating it violent cramps and diarrhea set inl3rd day sees a false remissionlBy 4th or 5th day death will occur unless a liver transp

17、lant is donelThe symptoms are due to inhibition of RNAPII and manifest mainly in liverThe chemical structure of -amanitin* All 3 are big, multimeric proteins (500-700 kD) * All have 2 large subunits with sequences similar to and in E.coli RNAP, so catalytic site may be conserved * All have subunit h

18、omologs of a in E. coli RNAP* However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli factor. * These features are shared by RNAPs across speciesRNA polymerases I, II and III have structural features in common: The subunits of yeast RNA polymerase IISubunitFunction

19、RPB1RPB2RPB3RPB4RPB5RPB6RPB7RPB8RPB9RPB10RPB11RPB12Binds DNA (and has CTD)Binds NTPsassemblyNot essential, (stress response)Target for activatorsEfficient assemblyNot essential(stress response)Not knownHelps select start siteNot knownAssemblyNot knownE. coli Homologb bb ba1a1w wa2a2Size (kD)22015045

20、322723171413101010(PNAS January 30, 2001 98, 892-897)RNAP core structure fromT. Aquaticus ）.RNAP II structure fromyeast.Comparison of RNAP structuresin karyotes and eukaryotesNote that the overall shape of the enzyme is the same.Also the overall positions of the subunit homologs are the same.Most in

21、teresting because it regulates synthesis of mRNA Yeast Pol II consists of 12 different subunits named according to size, from largest to smallest (RPB1 - RPB12) RPB1 and RPB2 are homologous to E. coli RNA polymerase and RPB1 has DNA-binding site; RPB2 binds NTP RPB1 and RPB2 together make up the act

22、ive siteRPB3 and RPB11 are homologs of a1 and a2Important feature of RPB1 * Although RPB1 is very similar to E. coli in sequence, structure and position in the enzyme, there is an important difference between the two subunits * RPB1 is longer at the carboxy terminal end * This extension of amino aci

23、d sequence at the carboxy-terminal end of RPB1 is called the CTD, which stands for C-terminal domain. * This domain is unique to the largest subunit of RNAP II.* It is NOT found in the related largest subunits of RNAP I or III, or in the E. coli RNAP largest subunitThe sequence of the CTD is an unus

24、ual 7 amino-acid repeat with an “extended” structure2652l therefore the CTD is essential for RNAP II functionl the CTD has a lot of S, T amino acids which can be phosophorylatedl it is known that RNAP II in an initiation complex has a non-phosphorylated CTDl deletion studies showed that yeast requir

25、e at least 13 repeats to survivel elongating RNAP II has a phosphorylated CTDRNA Polymerase II * RPB1 has C-terminal domain (CTD) or PTSPSYS* 5 of these 7 have -OH, so this is a hydrophilic and phosphorylatable site * CTD is essential and this domain may project away from the globular portion of the

26、 enzyme (up to 50 nm!) * Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription Viral RNA Polymerases* T7 RNA Polymerase* T3 RNA Polymerase* SP6 RNA PolymeraseFig. 1 Structure of transcribing T7RNA polymeraseThe T7 RNA polymerase has a structure similar to bacterial RNA polymeras

27、e and eukaryotic mitochondrial and chloroplast enzymes.Cheetham & Steitz (1999) Structure of a transcribing T7 RNA polymerase initiation complex, Science 286: 2305-2309.Structure of the transcribing RNA polymeraseDetailed Transcriptional Mechanism* Three-step process* 1) Initiation* 2) Elongatio

28、n* 3) Termination* DNA transcription in prokaryotes* DNA transcription in eukaryotes* In vitro DNA transcription* Initiation* 1) what is promoter?* 2) how to determine the promoter * sequences?-DNase I footprinting* 3) Consensus sequences* 4) Formation of transcriptional * complex* Elongation* Termi

29、nationRadio-labeling DNase I Partial DigestionMissing segmentsElectrophoresisAutoradiographyFootprinting-10 regionRNAP binds a region of DNA from -40 to +20The sequence of the non-template strand is shownTTGACA16-19 bp. TATAAT “-35” spacer “-10”Properties of Promoters * Promoters typically consist o

30、f 40 bp region on the 5-side of the transcription start site * Two consensus sequence elements: * The -35 region, with consensus TTGACA * The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding - why? * “UP element” (an AT-rich sequence about 20 bp in size located immedi

31、ately upstream of the -35 region;). The seven E. coli rrn genes, which encode ribosomal RNA, are unusually strong Important Promoter Features (tested by mutations) * The closer the match to the consensus, the stronger the* promoter (-10 and -35 boxes)* The absolute sequence of the spacer region (bet

32、ween the -10 * and -35 boxes) is not important* The length of the spacer sequence IS important:*TTGACA - spacer (16 to 19 base pairs) - TATAAT* Spacers that are longer or shorter than the consensus length* make weak promoters* Polymerase binds nonspecifically to DNA with low affinity and migrates, l

33、ooking for promoter * Sigma subunit recognizes promoter sequence * RNA polymerase holoenzyme and promoter form closed promoter complex (DNA not unwound) - Kd = 10-6 to 10-9 M * Polymerase unwinds about 12 pairs to form open promoter complex - Kd = 10-14 M * Binding of RNAP to Template DNA* RNA polym

34、erase has two binding sites for NTPs * Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5-end) * Elongation site binds the second incoming NTP * 3-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) * When 6-10 unit oligonucleotide ha

35、s been made, sigma subunit dissociates, completing initiation Transcriptional regulation in bacteria2. Interaction of RNA polymerase with templates ss sSigma leavesafter 10 nt aretranscribed.Sigma joinscomplexWhen does sigma leave?Is sigma still present after fingersclose, but before abortive cyclin

36、ghas stopped?Finding and binding the promoterClosed complex formationRNAP bound -40 to +20Open complex formationRNAP unwinds from -10 to +2Binding of 1st NTPRequires high purine NTPAddition of next NTPsRequires lower NTPsDissociation of sigmaAfter RNA chain is 6-10 NTPs longTranscriptional regulatio

37、n in bacteriaTranscription cycleFig. 9.9Abortive initiation or cycling:RNA pol transcribe 2-9 nt andthen restarts. Does not leavethe promoter. May occur severalhundred times before true elongation.Note: the number of bases that canbe packed into the active site of the enzyme is 8. This correlates wi

38、thabortive products of 8-10 bases.Transcriptional regulation in bacteriaFig. 9.9Sometimes RNA polymerase pausesdue to a temporary shortage of thecomplementary nucleotide. When thisoccurs, restarting synthesis requiresthe GreA and GreB proteins to releasethe pause. The 3 end is cleaved so that it is

39、properly aligned within thecatalytic site of the polymerase again.Core polymerase - no sigma factorPolymerase is pretty accurate - only about 1 error in 10,000 bases (not as accurate as DNAP III)Even this error rate is OK, since many transcripts are made from each gene Elongation rate is 20-50 bases

40、 per second - slower in G/C-rich regions and faster elsewhere Topoisomerases precede and follow polymerase to relieve super coiling Science, vol. 281, p 424 (1998)Spatial Organization of Transcription Elongation Complex in E. coliInteractions between nucleic acids and the core enzyme keep RNAP proce

41、ssiveTwo mechanisms Rho() - the termination factor protein rho is an ATP-dependent helicase it moves along RNA transcript, finds the bubble, unwinds it and releases RNA chain Specific sequences - termination sites in DNA inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript 6-8 As

42、in DNA coding for Us in transcript Chain Termination Rho-independent transcription termination (depends on DNA sequence - NOT a protein factor)Stem-loop structureRho-independent transcription termination RNAP pauses when it reaches a termination site. The pause may give the hairpin structure time to

43、 fold The fold disrupts important interactions between the RNAP and its RNA product The U-rich RNA can dissociate from the template The complex is now disrupted and elongation is terminatedRho-Dependent Transcription Termination(depends on a protein AND a DNA sequence)G/C -rich siteRNAP slows downRh

44、o helicase catches upElongating complex is disrupted* Multiple Polymerases at least 3 types of RNAPs* Chromatin and Nucleosomes* Unable to initiate transcription on their own - Require Transcription Factors (TF)* Unable to recognize Promoters on their own * Primary transcripts contain exons * The Pr

45、omoters are complex. Multiple regulatory proteins can bind to the promoter. * Cis-acting elements and Trans-acting factors.* Enhancer, silencer & insulator* mRNAs are mostly monocistronic * Genes controlled by positive control - off unless activators are present * In eukaryotes, transcription an

46、d translation occur in separate compartments.Enhancers can occur in a variety of positions with respect to genesTranscription unitPEx1Ex2EnhancerEnhancerAdjacentDownstreamInternalDistalUpstreamTrans-acting factorTranscription factor* ERE - Estrogen response element * HSE - Heat shock element * MRE -

47、 Metal response element * GRE - Glucocorticoid response element DNA transcription by RNAP I* Promoters* 1) Core promoter (-45 to +20)* 2) Upstream control element (UCE; -180 to -107)* Distance between UCE and promoter is critical Species-specific* TFs* 1) SL1: TBF and TAF* 2) UBF* Mechanism (1) Init

48、iation & Elongation* (2) termination-requires a specific DNA-binding termination factorUCECore PromoterStart-pointDNA transcription by RNAP III*Promoters* 1) Some Pol III genes (tRNA, 5S rRNA) have * internal promoters* 5S rRNA: Box A & Box C* tRNA: Box A & Box B* 2) Some are “pol II-lik

49、e”* Eg: some snRNAs have essential TATA * box and upstream promoter elements*TFIII* TFIIIA、B and C:* TFIIIC binds both A box and B box.* TFIIIB: TBP、BRF（TFIIIB-related factor， * and TFIIIB*Mechamism * (1) Initiation & Elongation* (2) Termination-terminates after U-series, but no apparent upstrea

50、m stem-loopA BoxB BoxStartpoint* Promoters-usually contain one or more of the following:* Initiator ,TATA box (Hogness box） * and Upstream element（UPE)* 1) Initiator (Inr): consensus sequence * PyPyANT/APyPy; (A is +1)* 2) TATA box* Consensus sequence TATAAAA* 3) Upstream elements* GC boxes (GGGCGG)

51、-Binding site for Sp1* CCAAT box -Binding site for CCAAT- * binding transcription factor (CTF) and * CCAAT/enhancer binding protein (C/EBP)* TF IIEnkaryotic PromoterThe core promoter located within about 30 bp of the start site An upstream promoter, which may extend over as many as 200 bp farther up

52、stream* The rows of lock boxes in a bank provide a useful analogy. * To open any particular box in the room requires two keys: * Your key, whose pattern of notches fits only the lock of the box assigned to you (= the upstream promoter), but which cannot unlock the box without * A key carried by a ba

53、nk employee that can activate the unlocking mechanism of any box (= the basal promoter) but cannot by itself open any box.RNAP I, II AND III PromotersPOL IIPOL IIIUPE -60TATA -30Inr +1A +20B +60UCE -90COREPSE -60TATA -30POL I eg AdMLeg tRNA(internal)eg tRNArRNABasal promoter for RNAP IIThe numbers under the