Bài giảng Molecular Biology - Chapter 6 The Mechanism of Transcription in Bacteria

Molecular BiologyFifth EditionChapter 6The Mechanism of Transcription in BacteriaLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.16.1 RNA Polymerase StructureBy 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits2 very large subunits are b (150 kD) and b’ (160 kD)Sigma (s) at 70 kDAlpha (a) at 40 kD – 2 copies present in holoenzymeOmega (w) at 10 kD Was not clearly visible in SDS-PAGE, but seen in other experimentsNot required for cell viability or in vivo enzyme activityAppears to play a role in enzyme assembly2Sigma as a Specificity FactorCore enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNAWith  subunit, the holoenzyme worked equally well on both types of DNA3SummaryThe key player in the transcription process is RNA polymeraseThe E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a -factor, which directs the core to transcribe specific genes46.2 PromotersWhy was the core RNA polymerase capable of transcribing nicked DNA in the previous table? Nicks and gaps are good sites for RNA polymerase to bind nonspecificallyThe presence of the s-subunit permits recognition of authentic RNA polymerase binding sites called promotersTranscription that begins at promoters is specific, directed by the s-subunit5Binding of RNA Polymerase to PromotersHow tightly does core enzyme v. holoenzyme bind DNA?Experiment measures binding of DNA to enzyme using nitrocellulose filtersHoloenzyme binds filters tightlyCore enzyme binding is more transient6Temperature and RNA Polymerase BindingAs the temperature is lowered, the binding of RNA polymerase to DNA decreases dramaticallyHigher temperatures promote DNA melting and encourage RNA polymerase binding7RNA Polymerase BindingHinkle and Chamberlin proposed:RNA polymerase holoenzyme binds DNA loosely at firstBinds at promoter initiallyScans along the DNA until it finds a promoterComplex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds formHoloenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA8Polymerase/Promoter BindingHoloenzyme binds DNA loosely at firstComplex loosely bound at promoter = closed promoter complex, dsDNA in closed formHoloenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound9SummaryThe -factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoterThis tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by The -factor can therefore select which genes will be transcribed10Core Promoter ElementsThere is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 boxAnother short sequence centered 35 bp upstream is known as the -35 boxComparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes11Promoter StrengthConsensus sequences:-10 box sequence approximates TATAAT-35 box sequence approximates TTGACAMutations that weaken promoter binding:Down mutationsIncrease deviation from the consensus sequenceMutations that strengthen promoter binding:Up mutationsDecrease deviation from the consensus sequence12UP ElementThe UP element is upstream of the core promoter, stimulating transcription by a factor of 30UP is associated with 3 “Fis” sites which are binding sites for the transcription-activator protein Fis, not for the polymerase itself13The rrnB P1 Promoter Transcription from the rrn promoters respond Positively to increased concentration of iNTPNegatively to the alarmone ppGpp146.3 Transcription InitiationTranscription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bondCarpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNAAbortive transcripts such as these have been found up to 10 nt15Stages of Transcription InitiationFormation of a closed promoter complexConversion of the closed promoter complex to an open promoter complexPolymerizing the early nucleotides – polymerase at the promoterPromoter clearance – transcript becomes long enough to form a stable hybrid with template16Sigma Stimulates Initiation of TranscriptionIn this first experiment stimulation by s appears to cause both initiation and elongationOr stimulating initiation by s provides more initiated chains for core polymerase to elongateFurther experiments by the same group proved that s does not stimulate elongation17Reuse of sDuring initiation s can be recycled for additional use with a new core polymeraseThe core enzyme can release s which is then free to associate with another core enzyme18Fluorescence Resonance Energy TransferThe -factor changes its relationship to the core polymerase during elongationIt may not dissociate from the core but actually shift position and become more loosely bound to coreTo answer this question Fluorescence Resonance Energy Transfer (FRET) was used as it relies on two fluorescent molecules that are close enough together to engage in transfer of resonance energyFRET allows the position of s relative to a site on the DNA to be measured without using separation techniques that might displace s from the core enzyme19FRET Assay for s Movement Relative to DNA20Models for the -CycleThe obligate release version of the -cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of  from core as polymerase undergoes promoter clearance and switches from initiation to elongation modeThe stochastic release model proposes that  is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model21Local DNA Melting at the PromoterFrom the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bpIn another experiment, the length of the melted region was found to be 12 bpLater, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp22Experiment to locate the region of early promoter melted by RNA Polymerase23Promoter ClearanceRNA polymerases have evolved to recognize and bind strongly to promotersThis poses a challenge when it comes time for promoter clearance as those strong bonds must be broken in order for polymerase to leave the promoter and enter the elongation phase24Promoter ClearanceSeveral hypotheses have been proposedThe polymerase cannot move enough downstream to make a 10-nt transcript without doing one of three things: - transient excursion: moving briefly downstream and then snapping back to the starting position - inchworming: stretching itself by leaving its trailing edge in place while moving ots leading edge downstream - scrunching: compressing the DNA without moving itself25Abortive Transcription, Scrunching and Promoter ClearanceEbert and colleagues performed several experiments to distinguish between the hypothesesUsing E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearanceThe E.coli polymerase achieves abortive transcription by scrunching: drawing downstream DNA into the polymerase without actually moving and losing its grip on promoter DNAThe scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription26Structure and Function of sGenes encoding a variety of s-factors have been cloned and sequencedThere are striking similarities in amino acid sequence clustered in 4 regionsConservation of sequence in these regions suggests important functionAll of the 4 sequences are involved in binding to core and DNA27Homologous Regions in Bacterial s Factors28E. coli s70Four regions of high sequence similarity are indicatedSpecific areas that recognize the core promoter elements are the -10 box and –35 box29Region 1Role of region 1 appears to be in preventing s from binding to DNA by itselfThis is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcriptionRegion 2This region is the most highly conserved of the fourThere are four subregions – 2.1 to 2.42.4 recognizes the promoter’s -10 boxThe 2.4 region appears to be a-helix30Regions 3 and 4Region 3 is involved in both core and DNA bindingRegion 4 is divided into 2 subregionsThis region seems to have a key role in promoter recognitionSubregion 4.2 contains a helix-turn-helix DNA-binding domain and appears to govern binding to the -35 box of the promoter31SummaryComparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sourcesSubregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognitionThe s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA-binding region of sRegion between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter32Role of a-Subunit in UP Element RecognitionRNA polymerase itself can recognize an upstream promoter element, UP elementWhile s-factor recognizes the core promoter elements, what recognizes the UP element?It appears to be the a-subunit of the core polymerase33Modeling the Function of the C-Terminal DomainRNA polymerase binds to a core promoter via its s-factor, no help from C-terminal domain of a-subunitBinds to a promoter with an UP element using s plus the a-subunit C-terminal domains (CTD)Results in very strong interaction between polymerase and promoterThis produces a high level of transcription346.4 ElongationAfter transcription initiation is accomplished, core polymerase continues to elongate the RNANucleotides are added sequentially, one after another in the process of elongation35Function of the Core PolymeraseCore polymerase contains the RNA synthesizing machineryPhosphodiester bond formation involves the b- and b’-subunitsThese subunits also participate in DNA bindingAssembly of the core polymerase is a major role of the a-subunit36Role of b in Phosphodiester Bond FormationCore subunit b lies near the active site of the RNA polymeraseThis active site is where the phosphodiester bonds are formed linking the nucleotidesThe s-factor may also be near the nucleotide-binding site during the initiation phase37Structure of the Elongation ComplexThis section will examine how well predictions have been borne out by structural studiesHow does the polymerase deal with problems of unwinding and rewinding templates?How does it move along the helical template without twisting RNA product around the template?38RNA-DNA HybridThe area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNAIn T7 the similar hybrid appears to be 8 bp long39Structure of the Core PolymeraseX-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab clawIt appears designed to grasp the DNAA channel through the enzyme includes the catalytic centerMg2+ ion coordinated by 3 Asp residuesRifampicin-binding site40Structure of the Holoenzyme-DNA ComplexCrystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals:DNA is bound mainly to -subunitInteractions between amino acids in region 2.4 of  and -10 box of promoter are possible3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted2 invariant basic amino acids in  predicted to function in DNA binding are positioned to do soA form of the polymerase that has 2 Mg2+ ions41Structure of the Elongation ComplexThe X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observationsa valine residue in the E’ subunit inserts into the minor groove of the downstream DNAthe downstream DNA is double-stranded up to and including the +2 bse pairthe enzyme can accommodate nine base pairs of RNA-DNA hybridthe RNA product in the exit channel is twisted into the shape it would assume as 1/2 of an A-form dsRNA42Topology of ElongationElongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNAPolymerase maintains a short melted region of template DNADNA must unwind ahead of the advancing polymerase and rewind behind itStrain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases43Pausing and ProofreadingRNA polymerase frequently pauses, or even backtracks, during elongationPausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in terminationBacktracking aids proofreading by extruding the 3’-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors446.5 Termination of TranscriptionWhen the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNAThere are 2 main types of terminatorsIntrinsic terminators function with the RNA polymerase by itself without help from other proteinsOther type depends on auxiliary factor called rho (r, these are rho or r-dependent terminators 45Rho-Independent TerminationIntrinsic or rho-independent termination depends on terminators of 2 elements:Inverted repeats followed immediately byT-rich region in the nontemplate strand of the geneAn inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences46Inverted Repeats and HairpinsThe repeat at right is symmetrical around its center shown with a dotA transcript of this sequence is self-complementaryBases can pair up to form a hairpin as seen in the lower panel 47Structure of an Intrinsic TerminatorAttenuator contains a DNA sequence that causes premature termination of transcriptionThe E. coli trp attenuator was used to show:Inverted repeat allows a hairpin to form at transcript endString of T’s in nontemplate strand result in weak rU-dA base pairs holding the transcript to the template strand48Model of Intrinsic TerminationBacterial terminators act by:Base-pairing of something to the transcript to destabilize RNA-DNA hybridCauses hairpin to formThis causes transcription to pausea string of U’s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template49Rho-Dependent TerminationRho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitroThis depression was due to termination of transcriptionAfter termination, polymerase must reinitiate to begin transcribing again50Rho Affects Chain ElongationThere is little effect of rho or r on transcription initiation, if anything it is increasedThe effect of rho or r on total RNA synthesis is a significant decreaseThis is consistent with action of rho or r to terminate transcription forcing time-consuming reinitiation51Rho Causes Production of Shorter TranscriptsSynthesis of much smaller RNAs occurs in the presence of rho or r compared to those made in the absenceTo ensure that this due to r itself and not to RNase activity of r, RNA was transcribed without r and then incubated in the presence of rThere was no loss of transcript size, so no RNase activity in r 52Rho Releases Transcripts from the DNA TemplateCompare the sedimentation of transcripts made in presence and absence of rWithout r, transcripts cosedimented with the DNA template – they hadn’t been releasedWith r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA templateIt appears that r serves to release the RNA transcripts from the DNA template53Mechanism of RhoNo string of T’s in the r-dependent terminator, just inverted repeat to hairpinBinding to the growing transcript, r follows the RNA polymeraseIt catches the polymerase as it pauses at the hairpinReleases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid54SummaryUsing the trp attenuator as a model rho-independet terminator revealed two important features: 1 - an inverted repeat that allows a hairpin to for at the end of the transcript 2 - a string of T’s in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strandRho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T’s55