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Lecture PowerPoint to accompany Molecular Biology Fourth 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 Testing Transcription • Core enzyme transcribes both DNA strands • Without s-subunit the core enzyme has basic transcribing ability but lacks specificity 6-4 6.2 Promoters • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • Presence of the s-subunit permitted recognition of authentic RNA polymerase binding sites • Polymerase binding sites are 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 temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperature promotes DNA melting 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 one • 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 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 for each of these boxes 6-10 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 6-11 UP Element • UP element is a promoter, stimulating transcription by a factor of 30 • UP is associated with 3 “Fis” sites which are binding sites for transcription-activator protein Fis, not for the polymerase itself • Transcription from the rrn promoters respond – Positively to increased concentration of iNTP – Negatively to the alarmone ppGpp 6-12 The rrnB P1 Promoter 6-13 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 6-14 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 6-15 The Functions of s • Gene selection for transcription by s causes tight binding between RNA polymerase and promoters • Tight binding depends on local melting of DNA that permits open promoter complex • Dissociation of s from core after sponsoring polymerase-promoter binding 6-16 Sigma Stimulates Transcription Initiation • Stimulation by s appears to cause both initiation and elongation • Or stimulating initiation provides more initiated chains for core polymerase to elongate 6-17 Reuse of s • During initiation s can be recycled for additional use in a process called the s cycle • Core enzyme can release s which then associates with another core enzyme 6-18 Sigma May Not Dissociate from Core During Elongation • The s-factor changes its relationship to the core polymerase during elongation • It may not dissociate from the core • May actually shift position and become more loosely bound to core 6-19 Fluorescence Resonance Energy Transfer • Fluorescence resonance energy transfer (FRET) relies on the fact that two fluorescent molecules close together will engage in transfer of resonance energy • FRET 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 enzyme 6-20 FRET Assay for s Movement Relative to DNA 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 Region of Early Promoter Melted by RNA Polymerase 6-23 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 6-24 Homologous Regions in Bacterial s Factors 6-25 E. coli s70 • Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements, -10 box and –35 box are notes 6-26 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 6-27 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 6-28 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 6-29 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 6-30 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 6-31 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 • Results in very strong interaction between polymerase and promoter • This produces a high level of transcription 6-32 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 6-33 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 6-34 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 nucleotide-binding site during initiation phase 6-35 Role of b’ and b in DNA Binding • In 1996, Evgeny Nudler and colleagues showed that both the b- and b’-subunits are involved in DNA binding • They also showed that 2 DNA binding sites are present – A relatively weak upstream site • DNA melting occurs • Electrostatic forces are predominant – Strong, downstream binding site where hydrophobic forces bind DNA and protein together 6-36 Strategy to Identify Template Requirements 6-37 Observations Relating to Polymerase Binding • Template transfer experiments have delineated two DNA sites that interact with polymerase • One site is weak – It involves the melted DNA zone, along with catalytic site on or near b-subunit of polymerase – Protein-DNA interactions here are mostly electrostatic and are salt-sensitive • Other is strong binding site involving DNA downstream of the active site and the enzyme’s b’- and b-subunits 6-38 Structure of the Elongation Complex • How 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? 6-39 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 6-40 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 6-41 Structure of the Holoenzyme • Crystal structure of T. aquaticus RNA polymerase holoenzyme shows an extensive interface between s and b- and b’-subunits of the core • Structure also predicts s region 1.1 helps open the main channel of the enzyme to admit dsDNA template to form the closed promoter complex • After 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 complex 6-42 Additional Holoenzyme Features • Linker joining s regions 3 and 4 lies in the RNA exit channel • As transcripts grow, they experience strong competition from s3-s4 linker for occupancy of the exit channel 6-43 Structure of the HoloenzymeDNA 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 6-44 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 close up behind it • Strain introduced into the template DNA is relaxed by topoisomerases 6-45 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 r, these are r-dependent terminators 6-46 Rho-Independent Termination • Intrinsic or r-independent termination depends on terminators of 2 elements: – Inverted repeat followed immediately by – T-rich region in nontemplate strand of the gene • An inverted repeat predisposes a transcript to form a hairpin structure 6-47 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 6-48 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 6-49 Model of Intrinsic Termination Bacterial terminators act by: • Base-pairing of something to the transcript to destabilize RNA-DNA hybrid – Causes hairpin to form • Causing the transcription to pause – Causes a string of U’s to be incorporated just downstream of hairpin 6-50 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 6-51 Rho Affects Chain Elongation • There is little effect of r on transcription initiation, if anything it is increased • The effect of r on total RNA synthesis is a significant decrease • This is consistent with action of r to terminate transcription forcing timeconsuming reinitiation 6-52 Rho Causes Production of Shorter Transcripts • Synthesis of much smaller RNAs occurs in the presence of r compared to those made in the absence • To ensure that this due to r, 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 6-53 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 6-54 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 6-55