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Outline“Central dogma”General properties of DNA transcriptionRNA polymeraseDetailed mechanisms of DNA transcription1.Bacterial DNA transcription2.Eukaryotic DNA transcription3.Archaeal DNA transcriptionTranscriptionTranslationReplicationReplicationRetro transcriptionGene expressionBasic features of DNA Transcription Common to DNA replication1. Template, Unwinding and Torsion-relieving are necessary; 2. Proceed only in the 53direction; Cordycepin can prove this Uncommon to DNA replication1. No need for primers2. NTPs instead of dNTPs; UTP instead of dTTP3. Lacking proof-reading activity ( error rate is 1 in 104 or 105 nts added )4. Specific regions (not all DNA sequence) can be transcribed5. To a specific gene, only one strand can be transcribedCoding strand, Sense strand, Crick strandTemplate strand, antisense strand, Watson strandTranscriptionTranslationGene expression and the related terminologyTranscription runs only in the 53direction; Cordycepin can prove thisBacterial RNA Polymerase * The first nucleic acid synthesizing enzyme ( polynucleotide phosphorylase, PNP ) In 1955, Marianne Grunberg-Manago and Severo Ochoa reported the isolation of an enzyme that catalyzed the synthesis of RNA from NDPs. 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 Hurwitz, A. Stevens and J. Bonner. This enzyme required a template, used all four NTPs as substrates and synthesized a product with a composition similar to that of the template, and it required Mg2+.DNA-Dependent RNA Polymerases (RNAP)* Common features -DNA template: one strand is copied -Substrate NTPs (GTP, CTP, UTP, ATP) -Divalent 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 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) 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 dNTPs10) RNAP incorporates UTP instead of dTTPStructure and Function of the Bacterial RNAPCAll three classes of RNAs are transcribed by the same RNA polymerase In E.coli, RNAP is 465 k complex, with 2, 1, 1, 1, 1 subunit *Holoenzyme*Core enzyme is 2, 1, 1, 1 *Inhibitors: Rifampicin & Streptolydigina aa a2 2 a a2 2b b a a2 2bbbb = core enzymea1bba2Core EnzymeSequence-independent,nonspecific transcriptioninitiation+vegetative(principal s) s s7070heat shock(for emergencies) s s3232nitrogen starvation(for emergencies) s s6060factorinterchangeable,promoter recognitionThe assembly pathway of the core enzyme(the subunit makes this more efficient)a1bba2s s7070RNAP HOLOENZYME - 70 Promoter-specific transcription initiationIn 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.* recognizes promoter sequences on DNAThe sigma factorCThe sigma subunit does two things:1.It reduces the affinity of the enzyme for non-specific DNA. 2.It greatly increases the affinity of the enzyme for promoters. E. coli also has six alternative sigma factors that are used in special circumstances 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 InhibitorN Mushrooms of the genus Amanita make a toxic cyclic octapeptide called a amanitin NThis mushroom tastes good but eating it is deadly!N6 to 24 hours after eating it violent cramps and diarrhea set inN3rd day sees a false remissionNBy 4th or 5th day death will occur unless a liver transplant is doneNThe symptoms are due to inhibition of RNAPII and manifest mainly in liverThe chemical structure of -amanitinRNAPs in Eukaryotes* All 3 are big, multimeric proteins (500-700 kD) * All have 2 large subunits with sequences similar to b b and b b in E.coli RNAP, so catalytic site may be conserved * All have subunit homologs 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: Comparison of RNAP structures in prokaryotes and eukaryotesFrom T. Aquaticusfrom yeastNote that the overall shape of the enzyme is the same.Also the overall positions of the subunit homologs are the same.The subunits of yeast RNA polymerase IIRNA Polymerase II Most interesting 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 b and b * RPB1 has DNA-binding site; RPB2 binds NTP * RPB1 and RPB2 together make up the active site RPB3 and RPB11 are homologs of 1 and 2Important features 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 acid 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 subunit*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 enzyme (up to 50 nm!) *Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription RNA Polymerase II The CTD has an unusual 7 amino-acid repeat with an “extended” structurethe CTD is essential for RNAP II functionthe CTD has a lot of S, T amino acids which can be phosophorylatedit is known that RNAP II in an initiation complex has a non-phosphorylated CTDdeletion studies showed that yeast require at least 13 repeats to surviveelongating RNAP II has a phosphorylated CTDBacterial 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* TerminationEMapping Promoters DNA sequences that guide RNAP to the start of a gene (transcription initiation site)Properties of Promoters * Promoters typically consist of 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 immediately upstream of the -35 region;). The seven E. coli rrn genes, which encode ribosomal RNA, are unusually strong The sense (coding) strand sequences of selected E. coli promotersThe structure of typical E. coli promotersImportance of the distance between the 35 and 10 regions of a promoterIncreased distance between 35 and 10 means they are no longer on the same side of the DNARNA polymerase is unable to contact both regionsOptimal spacing: RNA polymerase contacts both 35 and 10 regionsThe closer the match to the consensus, the stronger the promoter (-10 and -35 boxes) The absolute sequence of the spacer region (between 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 promotersImportant Promoter Features (tested by mutations) Binding of RNAP to Template DNA* Polymerase binds nonspecifically to DNA with low affinity and migrates, looking 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 Initiation of Transcription * Binding of RNAP to Template DNA* RNA polymerase 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 has been made, sigma subunit dissociates, completing initiation The initiation stage of transcription in bacteriaThe initiation stage of transcription in bacteria (continued)Transcriptional regulation 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 withabortive products of 8-10 bases.Transcriptional regulation in bacteriaFig. 9.9Sometimes due to a temporary shortage of thecomplementary nucleotide. When thisoccurs, restarting synthesis requiresthe and proteins to releasethe pause. The so that it is properly aligned within thecatalytic site of the polymerase again.Chain Elongation Core polymerase - no sigma factor*Polymerase 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 per second - slower in G/C-rich regions and faster elsewhere *Topoisomerases precede and follow polymerase to relieve super coiling Interactions between nucleic acids and the core enzyme keep RNAP processiveSynthesis of the RNA transcriptTwo mechanisms CRho() - the termination factor protein rho is an ATP-dependent helicase it moves along RNA transcript, finds the bubble, unwinds it and releases RNA chain CSpecific sequences - termination sites in DNA inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript 6-8 As in DNA coding for Us in transcript Chain Termination Structure of a typical terminatorRho-independent transcription terminationRho-dependent transcription terminationRho-dependent transcription termination (continued)Differences Transcription Bacteria vs. Eukaryotes*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 Promoters 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 *transcription and translation occur in separate compartments.Enhancers can occur in a variety of positions with respect to genesTranscription unitPEx1Ex2EnhancerEnhancerAdjacentDownstreamInternalDistalUpstreamTrans-acting factorTranscription factorOther Cis-acting Elements* ERE - Estrogen response element * HSE - Heat shock element * MRE - 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) Initiation & 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-like” 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 upstream stem-loopA BoxB BoxStartpointDNA transcription by RNAP II*Promoters-usually contain one or more of the following: Core promoter and Upstream elements*TF IIEnkaryotic RNAP II Core PromoterAn Analogy*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 bank employee that can activate the unlocking mechanism of any box (= the basal promoter) but cannot by itself open any box.The RNA polymerase II elongation complexThe general transcription factors for RNAP IIFormation of pre-initiation complexTBP (TFIID) binds to the promoter DNA (TATA + Inr)recruitsTFIIBrecruitsRNAP II + TFIIFrecruitTFIIErecruits and stimulatesTFIIHHelicase activity unwinds DNA near start siteKinase activity phosphorylates CTDFormation of pre-initiation complexAn elongation factor called TFIIS stimulates elongation for RNAP II. TFIIF also has a role in elongation.After termination of transcription the CTD is dephosphorylated and the RNAP II can re-enter a pre-initiation complex (PIC)Elongation by RNAP IIPOL IIIIFCTDMULTI-STEP MODELPRE-INITIATION COMPLEXINRTATAUPE-20+1IIDIIAIIBHJEMULTI-STEP MODELINITIATION COMPLEXPHOSPHORYLATION OF CTDINRTATAUPE-20+1IIDIIAIIBHJEPOL IIIIFCTDP P P PATPMULTI-STEP MODELPROMOTER CLEARANCEINRTATAUPE-20+1IIDIIAIIBHJEPOL IICTDP P P PRIBONUCLEOTIDESpre-mRNAELONGATIONFACTORSINITIATIONPROMOTER CLEARANCEELONGATIONTranscription in Archaea Transcription in the Archaea is similar to and distinct from what is observed in Bacteria and eucaryotes. Each archaeon has a single RNA polymerase responsible for transcribing all genes in the cell (as in Bacteria ). However, the RNA polymerase is larger and contains more subunits, many of which are similar to subunits in RNA polymerase II of eucaryotes. The promoters of archaeal genes are similar to those of eucaryotes in having a TATA box; binding of the archaeal RNA polymerase to its promoter requires a TATA-binding protein, just as in eucaryotes. Like the eucaryotic counterpart, the archaeal RNA polymerase also needs several additional transcription factors to function properly. Furthermore, some archaeal genes have introns, which must be removed by posttranscriptional processing. Finally, the mRNA molecules produced by transcription in Archaea are usually polycistronic, as in Bacteria. Promoter architecture and transcription in Archaea
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