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 1 st 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 –Mg 2+ 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 Mg 2+ 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