Molecular biology - Chapter 6: The mechanism of transcription in bacteria

Molecular BiologyFourth 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 s subunit, the holoenzyme worked equally well on both types of DNA3Testing TranscriptionCore enzyme transcribes both DNA strandsWithout s-subunit the core enzyme has basic transcribing ability but lacks specificity46.2 PromotersNicks and gaps are good sites for RNA polymerase to bind nonspecificallyPresence of the s-subunit permitted recognition of authentic RNA polymerase binding sitesPolymerase binding sites are 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 temperature is lowered, the binding of RNA polymerase to DNA decreases dramaticallyHigher temperature promotes DNA melting7RNA Polymerase BindingHinkle and Chamberlin proposed:RNA polymerase holoenzyme binds DNA loosely at firstBinds at promoter initiallyScans along the DNA until it finds oneComplex 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 bound9Core 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 for each of these boxes10Promoter 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 sequence11UP ElementUP element is a promoter, stimulating transcription by a factor of 30UP is associated with 3 “Fis” sites which are binding sites for transcription-activator protein Fis, not for the polymerase itselfTranscription from the rrn promoters respond Positively to increased concentration of iNTPNegatively to the alarmone ppGpp12The rrnB P1 Promoter136.3 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 nt14Stages 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 template15The Functions of sGene selection for transcription by s causes tight binding between RNA polymerase and promotersTight binding depends on local melting of DNA that permits open promoter complexDissociation of s from core after sponsoring polymerase-promoter binding16Sigma Stimulates Transcription InitiationStimulation by s appears to cause both initiation and elongationOr stimulating initiation provides more initiated chains for core polymerase to elongate17Reuse of sDuring initiation s can be recycled for additional use in a process called the s cycleCore enzyme can release s which then associates with another core enzyme18Sigma May Not Dissociate from Core During ElongationThe s-factor changes its relationship to the core polymerase during elongationIt may not dissociate from the coreMay actually shift position and become more loosely bound to core19Fluorescence Resonance Energy TransferFluorescence resonance energy transfer (FRET) relies on the fact that two fluorescent molecules close together will engage in transfer of resonance energyFRET allows the position of s relative to a site on the DNA to be measured with using separation techniques that might displace s from the core enzyme20FRET Assay for s Movement Relative to DNA21Local 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 bp22Region of Early Promoter Melted by RNA Polymerase23Structure 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 DNA24Homologous Regions in Bacterial s Factors25E. coli s70Four regions of high sequence similarity are indicatedSpecific areas that recognize the core promoter elements, -10 box and –35 box are notes26Region 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 transcription27Region 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-helix28Regions 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 promoter29SummaryComparison 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 promoter30Role 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 polymerase31Modeling 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 domainsResults in very strong interaction between polymerase and promoterThis produces a high level of transcription326.4 ElongationAfter transcription initiation is accomplished, core polymerase continues to elongate the RNANucleotides are added sequentially, one after another in the process of elongation33Function 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-subunit34Role 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 nucleotide-binding site during initiation phase35Role of b’ and b in DNA BindingIn 1996, Evgeny Nudler and colleagues showed that both the b- and b’-subunits are involved in DNA bindingThey also showed that 2 DNA binding sites are presentA relatively weak upstream siteDNA melting occursElectrostatic forces are predominantStrong, downstream binding site where hydrophobic forces bind DNA and protein together36Strategy to Identify Template Requirements37Observations Relating to Polymerase BindingTemplate transfer experiments have delineated two DNA sites that interact with polymeraseOne site is weakIt involves the melted DNA zone, along with catalytic site on or near b-subunit of polymeraseProtein-DNA interactions here are mostly electrostatic and are salt-sensitiveOther is strong binding site involving DNA downstream of the active site and the enzyme’s b’- and b-subunits38Structure of the Elongation ComplexHow do structural studies compare with functional studies of the core polymerase subunits?How 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?39RNA-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 long40Structure 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 site41Structure of the HoloenzymeCrystal structure of T. aquaticus RNA polymerase holoenzyme shows an extensive interface between s and b- and b’-subunits of the coreStructure also predicts s region 1.1 helps open the main channel of the enzyme to admit dsDNA template to form the closed promoter complexAfter helping to open channel, the s will be expelled from the main channel as the channel narrows around the melted DNA of the open promoter complex42Additional Holoenzyme FeaturesLinker joining s regions 3 and 4 lies in the RNA exit channelAs transcripts grow, they experience strong competition from s3-s4 linker for occupancy of the exit channel43Structure of the Holoenzyme-DNA ComplexCrystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals:DNA is bound mainly to s-subunitInteractions between amino acids in region 2.4 of s 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 s predicted to function in DNA binding are positioned to do soA form of the polymerase that has 2 Mg2+ ions44Topology 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 close up behind itStrain introduced into the template DNA is relaxed by topoisomerases456.5 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 r, these are r-dependent terminators 46Rho-Independent TerminationIntrinsic or r-independent termination depends on terminators of 2 elements:Inverted repeat followed immediately byT-rich region in nontemplate strand of the geneAn inverted repeat predisposes a transcript to form a hairpin structure47Inverted 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 48Structure 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 strand49Model of Intrinsic TerminationBacterial terminators act by:Base-pairing of something to the transcript to destabilize RNA-DNA hybridCauses hairpin to formCausing the transcription to pauseCauses a string of U’s to be incorporated just downstream of hairpin50Rho-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 again51Rho Affects Chain ElongationThere is little effect of r on transcription initiation, if anything it is increasedThe effect of r on total RNA synthesis is a significant decreaseThis is consistent with action of r to terminate transcription forcing time-consuming reinitiation52Rho Causes Production of Shorter TranscriptsSynthesis of much smaller RNAs occurs in the presence of r compared to those made in the absenceTo ensure that this due to r, 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 53Rho 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 template54Mechanism 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 hybrid55