Mechanism of transcription in bacteria.ppt
- 1. Molecular Biology Fourth Edition Chapter 6 The Mechanism of Transcription in Bacteria Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 2. 6-2 6.1 RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits – 2 very large subunits are (150 kD) and ’ (160 kD) – Sigma () at 70 kD – Alpha () 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
- 3. 6-3 Sigma as a Specificity Factor • Core enzyme without the 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
- 4. 6-4 Testing Transcription • Core enzyme transcribes both DNA strands • Without s-subunit the core enzyme has basic transcribing ability but lacks specificity
- 5. 6-5 6.2 Promoters • Nicks and gaps are good sites for RNA polymerase to bind nonspecifically • Presence of the -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 -subunit
- 6. 6-6 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
- 7. 6-7 Temperature and RNA Polymerase Binding • As temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically • Higher temperature promotes DNA melting
- 8. 6-8 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
- 9. 6-9 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
- 10. 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 for each of these boxes
- 11. 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
- 12. 6-12 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
- 13. 6-13 The rrnB P1 Promoter
- 14. 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
- 15. 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
- 16. 6-16 The Functions of • Gene selection for transcription by causes tight binding between RNA polymerase and promoters • Tight binding depends on local melting of DNA that permits open promoter complex • Dissociation of from core after sponsoring polymerase-promoter binding
- 17. 6-17 Sigma Stimulates Transcription Initiation • Stimulation by appears to cause both initiation and elongation • Or stimulating initiation provides more initiated chains for core polymerase to elongate
- 18. 6-18 Reuse of • During initiation can be recycled for additional use in a process called the cycle • Core enzyme can release which then associates with another core enzyme
- 19. 6-19 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
- 20. 6-20 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 relative to a site on the DNA to be measured with using separation techniques that might displace from the core enzyme
- 21. 6-21 FRET Assay for Movement Relative to DNA
- 22. 6-22 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
- 23. 6-23 Region of Early Promoter Melted by RNA Polymerase
- 24. 6-24 Structure and Function of • Genes encoding a variety of -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
- 25. 6-25 Homologous Regions in Bacterial Factors
- 26. 6-26 E. coli 70 • Four regions of high sequence similarity are indicated • Specific areas that recognize the core promoter elements, -10 box and –35 box are notes
- 27. 6-27 Region 1 • Role of region 1 appears to be in preventing from binding to DNA by itself • This is important as binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription
- 28. 6-28 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 -helix
- 29. 6-29 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
- 30. 6-30 Summary • Comparison of different 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 -factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA- binding region of • Region between amino acids 262 and 309 of ’ stimulates binding to the nontemplate strand in the -10 region of the promoter
- 31. 6-31 Role of -Subunit in UP Element Recognition • RNA polymerase itself can recognize an upstream promoter element, UP element • While -factor recognizes the core promoter elements, what recognizes the UP element? • It appears to be the -subunit of the core polymerase
- 32. 6-32 Modeling the Function of the C- Terminal Domain • RNA polymerase binds to a core promoter via its - factor, no help from C- terminal domain of -subunit • Binds to a promoter with an UP element using plus the -subunit C-terminal domains • Results in very strong interaction between polymerase and promoter • This produces a high level of transcription
- 33. 6-33 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
- 34. 6-34 Function of the Core Polymerase • Core polymerase contains the RNA synthesizing machinery • Phosphodiester bond formation involves the - and ’-subunits • These subunits also participate in DNA binding • Assembly of the core polymerase is a major role of the -subunit
- 35. 6-35 Role of in Phosphodiester Bond Formation • Core subunit lies near the active site of the RNA polymerase • This active site is where the phosphodiester bonds are formed linking the nucleotides • The -factor may also be near nucleotide-binding site during initiation phase
- 36. 6-36 Role of ’ and in DNA Binding • In 1996, Evgeny Nudler and colleagues showed that both the - and ’-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
- 37. 6-37 Strategy to Identify Template Requirements
- 38. 6-38 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 -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 ’- and -subunits
- 39. 6-39 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?
- 40. 6-40 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
- 41. 6-41 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
- 42. 6-42 Structure of the Holoenzyme • Crystal structure of T. aquaticus RNA polymerase holoenzyme shows an extensive interface between and - and ’-subunits of the core • Structure also predicts 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
- 43. 6-43 Additional Holoenzyme Features • Linker joining regions 3 and 4 lies in the RNA exit channel • As transcripts grow, they experience strong competition from 3-4 linker for occupancy of the exit channel
- 44. 6-44 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
- 45. 6-45 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
- 46. 6-46 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 , these are -dependent terminators
- 47. 6-47 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
- 48. 6-48 Inverted Repeats and Hairpins • The repeat at right is symmetrical around its center shown with a dot • A transcript of this sequence is self- complementary – Bases can pair up to form a hairpin as seen in the lower panel
- 49. 6-49 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
- 50. 6-50 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
- 51. 6-51 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
- 52. 6-52 Rho Affects Chain Elongation • There is little effect of on transcription initiation, if anything it is increased • The effect of on total RNA synthesis is a significant decrease • This is consistent with action of to terminate transcription forcing time- consuming reinitiation
- 53. 6-53 Rho Causes Production of Shorter Transcripts • Synthesis of much smaller RNAs occurs in the presence of compared to those made in the absence • To ensure that this due to , not to RNase activity of , RNA was transcribed without and then incubated in the presence of • There was no loss of transcript size, so no RNase activity in
- 54. 6-54 Rho Releases Transcripts from the DNA Template • Compare the sedimentation of transcripts made in presence and absence of – Without , transcripts cosedimented with the DNA template – they hadn’t been released – With present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template • It appears that serves to release the RNA transcripts from the DNA template
- 55. 6-55 Mechanism of Rho • No string of T’s in the - dependent terminator, just inverted repeat to hairpin • Binding to the growing transcript, 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