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Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 6 The Mechanism of Transcription in Bacteria Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits – 2 very large subunits are b (150 kD) and b’ (160 kD) – Sigma (s) at 70 kD – Alpha (a) at 40 kD – 2 copies present in holoenzyme – Omega (w) at 10 kD • Was not clearly visible in SDS-PAGE, but seen in other experiments • Not required for cell viability or in vivo enzyme activity • Appears to play a role in enzyme assembly 6-2 Sigma as a Specificity Factor • Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA • With s subunit, the holoenzyme worked equally well on both types of DNA 6-3 Summary • The key player in the transcription process is RNA polymerase • The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a s-factor, which directs the core to transcribe specific genes 6-4 6.2 Promoters • Why 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 nonspecifically • The presence of the s-subunit permits recognition of authentic RNA polymerase binding sites called promoters • Transcription that begins at promoters is specific, directed by the s-subunit 6-5 Binding of RNA Polymerase to Promoters • How tightly does core enzyme v. holoenzyme bind DNA? • Experiment measures binding of DNA to enzyme using nitrocellulose filters – Holoenzyme binds filters tightly – Core enzyme binding is more transient 6-6 Temperature and RNA Polymerase Binding • As the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperatures promote DNA melting and encourage RNA polymerase binding 6-7 RNA Polymerase Binding Hinkle and Chamberlin proposed: • RNA polymerase holoenzyme binds DNA loosely at first – Binds at promoter initially – Scans along the DNA until it finds a promoter • Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form • Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA 6-8 Polymerase/Promoter Binding • Holoenzyme binds DNA loosely at first • Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form • Holoenzyme melts DNA at promoter forming open promoter complex polymerase tightly bound 6-9 Summary • The s-factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter • This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by s • The s-factor can therefore select which genes will be transcribed 6-10 Core Promoter Elements • There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box • Another short sequence centered 35 bp upstream is known as the -35 box • Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes 6-11 Promoter Strength • Consensus sequences: – -10 box sequence approximates TATAAT – -35 box sequence approximates TTGACA • Mutations that weaken promoter binding: – Down mutations – Increase deviation from the consensus sequence • Mutations that strengthen promoter binding: – Up mutations – Decrease deviation from the consensus sequence 6-12 UP Element • The UP element is upstream of the core promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for the transcriptionactivator protein Fis, not for the polymerase itself 6-13 The rrnB P1 Promoter • Transcription from the rrn promoters respond –Positively to increased concentration of iNTP –Negatively to the alarmone ppGpp 6-14 6.3 Transcription Initiation • Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond • Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA • Abortive transcripts such as these have been found up to 10 nt 6-15 Stages of Transcription Initiation • Formation of a closed promoter complex • Conversion of the closed promoter complex to an open promoter complex • Polymerizing the early nucleotides – polymerase at the promoter • Promoter clearance – transcript becomes long enough to form a stable hybrid with template 6-16 Sigma Stimulates Initiation of Transcription • In this first experiment stimulation by s appears to cause both initiation and elongation • Or stimulating initiation by s provides more initiated chains for core polymerase to elongate • Further experiments by the same group proved that s does not stimulate elongation 6-17 Reuse of s • During initiation s can be recycled for additional use with a new core polymerase • The core enzyme can release s which is then free to associate with another core enzyme 6-18 Fluorescence Resonance Energy Transfer • The s-factor changes its relationship to the core polymerase during elongation • It may not dissociate from the core but actually shift position and become more loosely bound to core • To 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 energy • FRET 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 enzyme 6-19 FRET Assay for s Movement Relative to DNA 6-20 Models for the s-Cycle • The obligate release version of the s-cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of s from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode • The stochastic release model proposes that s 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 model 6-21 Local DNA Melting at the Promoter • From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp • In another experiment, the length of the melted region was found to be 12 bp • Later, size of the DNA transcription bubble in complexes where transcription was active was found to be 17-18 bp 6-22 Experiment to locate the region of early promoter melted by RNA Polymerase 6-23 Promoter Clearance • RNA polymerases have evolved to recognize and bind strongly to promoters • This 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 phase 6-24 Promoter Clearance • Several hypotheses have been proposed • The 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 itself 6-25 Abortive Transcription, Scrunching and Promoter Clearance • Ebert and colleagues performed several experiments to distinguish between the hypotheses • Using E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearance • The E.coli polymerase achieves abortive transcription by scrunching: drawing downstream DNA into the polymerase without actually moving and losing its grip on promoter DNA • The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the 6-26 promoter and begin productive transcription Structure and Function of s • Genes encoding a variety of s-factors have been cloned and sequenced • There are striking similarities in amino acid sequence clustered in 4 regions • Conservation of sequence in these regions suggests important function • All of the 4 sequences are involved in binding to core and DNA 6-27 Homologous Regions in Bacterial s Factors 6-28 E. coli s70 • Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements are the -10 box and – 35 box 6-29 Region 1 • Role of region 1 appears to be in preventing s from binding to DNA by itself • This is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription Region 2 • • • • This region is the most highly conserved of the four There are four subregions – 2.1 to 2.4 2.4 recognizes the promoter’s -10 box The 2.4 region appears to be a-helix 6-30 Regions 3 and 4 • Region 3 is involved in both core and DNA binding • Region 4 is divided into 2 subregions – This region seems to have a key role in promoter recognition – Subregion 4.2 contains a helix-turn-helix DNA-binding domain and appears to govern binding to the -35 box of the promoter 6-31 Summary • Comparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sources • Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition • The s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNAbinding region of s • Region between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter 6-32 Role of a-Subunit in UP Element Recognition • RNA polymerase itself can recognize an upstream promoter element, UP element • While s-factor recognizes the core promoter elements, what recognizes the UP element? • It appears to be the a-subunit of the core polymerase 6-33 Modeling the Function of the CTerminal Domain • RNA polymerase binds to a core promoter via its s-factor, no help from C-terminal domain of a-subunit • Binds 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 promoter • This produces a high level of transcription 6-34 6.4 Elongation • After transcription initiation is accomplished, core polymerase continues to elongate the RNA • Nucleotides are added sequentially, one after another in the process of elongation 6-35 Function of the Core Polymerase • Core polymerase contains the RNA synthesizing machinery • Phosphodiester bond formation involves the b- and b’-subunits • These subunits also participate in DNA binding • Assembly of the core polymerase is a major role of the a-subunit 6-36 Role of b in Phosphodiester Bond Formation • Core subunit b lies near the active site of the RNA polymerase • This active site is where the phosphodiester bonds are formed linking the nucleotides • The s-factor may also be near the nucleotide-binding site during the initiation phase 6-37 Structure of the Elongation Complex • This section will examine how well predictions have been borne out by structural studies • 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? 6-38 RNA-DNA Hybrid • The 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 RNA • In T7 the similar hybrid appears to be 8 bp long 6-39 Structure of the Core Polymerase • X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw • It appears designed to grasp the DNA • A channel through the enzyme includes the catalytic center – Mg2+ ion coordinated by 3 Asp residues – Rifampicin-binding site 6-40 Structure of the Holoenzyme-DNA Complex Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals: – DNA is bound mainly to s-subunit – Interactions between amino acids in region 2.4 of s and -10 box of promoter are possible – 3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted – 2 invariant basic amino acids in s predicted to function in DNA binding are positioned to do so – A form of the polymerase that has 2 Mg2+ ions 6-41 Structure of the Elongation Complex • The X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observations – a valine residue in the E’ subunit inserts into the minor groove of the downstream DNA – the downstream DNA is double-stranded up to and including the +2 bse pair – the enzyme can accommodate nine base pairs of RNA-DNA hybrid – the RNA product in the exit channel is twisted into the shape it would assume as 1/2 of an Aform dsRNA 6-42 Topology of Elongation • Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA • Polymerase maintains a short melted region of template DNA • DNA must unwind ahead of the advancing polymerase and rewind behind it • Strain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases 6-43 Pausing and Proofreading • RNA polymerase frequently pauses, or even backtracks, during elongation • Pausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in termination • Backtracking 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 factors 6-44 6.5 Termination of Transcription • When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA • There are 2 main types of terminators – Intrinsic terminators function with the RNA polymerase by itself without help from other proteins – Other type depends on auxiliary factor called rho (r, these are rho or r-dependent terminators 6-45 Rho-Independent Termination • Intrinsic or rho-independent termination depends on terminators of 2 elements: – Inverted repeats followed immediately by – T-rich region in the nontemplate strand of the gene • An inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences 6-46 Inverted Repeats and Hairpins • The repeat at right is symmetrical around its center shown with a dot • A transcript of this sequence is selfcomplementary – Bases can pair up to form a hairpin as seen in the lower panel 6-47 Structure of an Intrinsic Terminator • Attenuator contains a DNA sequence that causes premature termination of transcription • The E. coli trp attenuator was used to show: – Inverted repeat allows a hairpin to form at transcript end – String of T’s in nontemplate strand result in weak rU-dA base pairs holding the transcript to the template strand 6-48 Model of Intrinsic Termination Bacterial terminators act by: • Base-pairing of something to the transcript to destabilize RNA-DNA hybrid – Causes hairpin to form • This causes transcription to pause – a string of U’s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template 6-49 Rho-Dependent Termination • Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro • This depression was due to termination of transcription • After termination, polymerase must reinitiate to begin transcribing again 6-50 Rho Affects Chain Elongation • There is little effect of rho or r on transcription initiation, if anything it is increased • The effect of rho or r on total RNA synthesis is a significant decrease • This is consistent with action of rho or r to terminate transcription forcing timeconsuming reinitiation 6-51 Rho Causes Production of Shorter Transcripts • Synthesis of much smaller RNAs occurs in the presence of rho or r compared to those made in the absence • To 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 r • There was no loss of transcript size, so no RNase activity in r 6-52 Rho Releases Transcripts from the DNA Template • Compare the sedimentation of transcripts made in presence and absence of r – Without r, transcripts cosedimented with the DNA template – they hadn’t been released – With r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template • It appears that r serves to release the RNA transcripts from the DNA template 6-53 Mechanism of Rho • No string of T’s in the rdependent terminator, just inverted repeat to hairpin • Binding to the growing transcript, r follows the RNA polymerase • It catches the polymerase as it pauses at the hairpin • Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid 6-54 Summary • Using 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 strand • Rho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T’s 6-55