Ch. 6 Mechanism of Transcription in Bacteria (not Archaea)

1. Ch. 6 Mechanism of Transcription in Bacteria(not Archaea) Student learning outcomes: • Explain that the core RNA polymerase (RNAP) consists of multiple subunits • Explain that sigma specificity factor chooses promoter • Explain the basic features of promoter sequences • Explain the nature of terminators: intrinsic (rho-independent) and rho-dependent • Appreciate how structural analysis have aided molecular mechanisms of understanding

2. Overview of bacterial transcription: • RNA polymerase (RNAP) + sigma (s) factor bind promoter sequences (closed complex RPc) • RNAP locally melts 10-17 bp of DNA (open RPo) • Initiation of transcription (first few nucleotides) • Elongation of transcription • Termination and release of transcript • Important Figures: 1, 3, 5, 6*, 9*, 12, 13, 16, 17, 19, 20, 29, 30, 34, 35, 38, 43, 44 • Review questions: 1, 6, 7, 9, 14, 17, 18, 19, 23, 24, 27, 28, 33, 34; Analyt Q 1, 2, 3

3. Basic gene structure; transcription start is +1 Fig. 3.20

4. 6.1 RNA Polymerase Structure s 5 4 3 2 1 holo SDS-PAGE of RNA polymerase (RNAP) from E. coli several subunits: • b (150 kD) and b’ (160 kD) • Sigma (s) at 70 kD • Alpha (a) at 40 kD – 2 copies present • Omega (w) at 10 kD • Not required for cell viability or in vivo enzyme activity • role in enzyme assembly Fig. 1 Purifications RNAP Pcellulose; Fr A, B, C

5. Sigma is a Specificity Factor • Core enzyme (without s subunit) did not transcribe viral DNA, yet did transcribe nicked calf thymus DNA; • Core Transcribes both strands (Fig. 2) • With s subunit, holoenzyme worked equally well on both types of DNA

6. 6.2 Promoters • Nicks and gaps - sites RNAP binds nonspecifically • The s-subunit permits recognition of authentic RNAP binding sites • RNAP binding sites are promoters • Transcription from promoters is specific, directed by s-subunit

7. RNA Polymerase Binds to Promoters • s stimulates tight binding of RNAP to promoter DNA • Measured binding of T7 DNA to RNAP using nitrocellulose filters • Protein sticks to filter, plus DNA bound to it; • At to, add excess unlabeled DNA, replaces labeled if RNAP falls off • Holoenzyme binds DNA tightly • Core enzyme binding is weak Fig. 3

8. Temperature and RNAP Binding to promoter • Form complexes, add lots unlabeled DNA • At lower temperatures, binding of RNAP to T7 DNA is decreased • Higher temperature promotes DNA melting -> stronger complexes Fig. 4

9. Polymerase/Promoter Binding: RPc -> RPo Hinkle & Chamberlin Holoenzyme binds DNA loosely at first Complex loosely bound at promoter = closed promoter complex (RPc),dsDNA closed form Holoenzyme melts DNA at promoter forming open promoter complex – (Rpo) polymerase tightly bound Fig. 5

10. Core Promoter Elements are conserved • Region common to bacterial promoters 6-7 bp long, 10 bp upstream of transcription start (+1) = -10 box • Sequence centered 35 bp upstream is -35 box • Comparison of thousands of promoters gave consensus sequence for each of these boxes • (capital letters >50%; lower case <50%) Fig. 6

11. Promoter Strength: transcription amount; reflects RNAP binding • Consensus sequences: • -10 box sequence approximates TAtAaT • -35 box sequence approximates TTGACa • Start of transcription is defined as +1 • Mutations that weaken promoter : • Down mutations • Increase deviation from consensus sequence • Mutations that strengthen promoter: • Up mutations • Decrease deviation from consensus sequence

12. Very strong promoters have UP Elementex. Promoter for rRNA gene • UP element (-40 to -60) stimulates transcription 30X; binds RNAP • UP region also 3 binding sites for transcription-activator protein Fis, (-60 to -150; an enhancer) • Transcription from these ribosomal rrn promoters responds to nucleotides (conc. iNTP) Fig. 7; rrnB P1 promoter

13. 1 2 3---- 6.3 Transcription Initiation • Initiation assumed to end as RNA polymerase formed 1st phosphodiester bond • Carpousis and Gralla found very small oligonucleotides (2-6 nt long) made without RNAP leaving DNA • Abortive transcripts up to 10 nt Fig. 8; E. coli RNAP; lane 1 no promoter; lane 2 [32P]ATP only; other lanes all nucleotides, inc.

14. Stages of Transcription Initiation • Formation of closed promoter complex (RPc) • Conversion of closed promoter complex to open promoter complex (RPo) • RNAP at promoter -polymerizing early nucleotides • Promoter clearance – transcript long enough to form stable hybrid with template • Factor s leaves

15. Recall RNA transcripts initiate with NTP (triphosphate); • 1st nucleotide has g phosphate; • phosphodiester bonds have only a phosphate Fig. 3.13

16. Sigma Stimulates Initiation • Stimulation by sappeared to cause both initiation and elongation • However, stimulating initiation provides more initiated chains for core polymerase to elongate • Later expts with rifampicin to block re-initiation showed not elongation Fig. 10. T4 DNA; [14C]ATP measures bulk RNA; [g -32P]NTP is initiation (most start A)

17. Reuse of s Figs. 11 and 12 • During initiation s recycled for additional use in process called the s cycle • Core enzyme can release s; associates with another core enzyme • Red [g -32P]ATP; then RifR core + Rif (green) or –Rif (blue)

18. Sigma May Not Actually Dissociate from Core RNAP During Elongation • Sigma s-factor changes its relationship to core RNAP during elongation • It may not actually dissociate from core • It may shift position and become more loosely bound • FRET (Fluorescence resonance energy transfer): two fluorescent molecules close together will transfer resonance energy FRET permits measurement of position of s relative to site on DNA without using separation techniques that might displace s from core RNAP (Ebright and colleagues)

19. FRET Assay for s Movement Relative to DNA Fig. 13 Predictions FRET. Fig. 14 FRET expt suggests sigma does not actually dissociate from RNAP

20. Local DNA Melts at Promoter • From number of RNAP holoenzymes bound to DNA, calculate each polymerase caused melting of about 10 bp • In another experiment, length of melted region was about12 bp • Size of DNA transcription bubble in complexes with active transcription was17-18 bp • Transcription bubble moves with RNAP, exposing template strand

21. Locate region of promoter melted by RNAP: DMS treatment of phage T7 Early Promoter: -9 to +3 Figs. 16, 17: Dimethyl sulfate methylation of DNA prevents base pairs reforming, renders melted region sensitive to nuclease S1. R = RNAP, S = S1

22. Structure and Function of s • Genes encoding variety of s-factors cloned and sequenced • Striking similarities in amino acid sequences - clustered in 4 regions • Conserved sequences suggest important function • All 4 sequences involved in binding RNAP and DNA • Primary sigmas (routine work): of E. coli = s70 of Bacillus subtilis = s43 (masses kD)

23. Homologous Regions in Bacterial s Factors Fig. 19 E. Coli and B. subtilis s factors

24. E. colis70 • Specific areas recognize core promoter elements: -10 box and –35 box • Region 1: prevents s from binding DNA without RNAP • Region 2: very conserved (subregion 2.4 recognizes promoter’s -10 box; alpha helix structure) • Region 3: both RNAP and DNA binding • Region 4: 2 subregions, key role in promoter recognition. subregion 4.2 has helix-turn-helix DNA-binding domain binds -35 box of promoter

25. Summary of s and RNAP • Comparison of different s gene sequences reveals 4 regions of similarity among variety of sources • Subregions 2.4 and 4.2 are involved in promoter; • -10 box and -35 box recognition • s-factor alone cannot bind DNA, but DNA interaction with core RNAP unmasks DNA-binding region of s • RNAP region between amino acids 262 and 309 of b’ stimulates s binding to nontemplate strand in -10 region of the promoter

26. C-Terminal Domain of a subunit of RNAP can recognize UP element • RNA polymerase binds core promoter via s-factor, no help from C-terminal domain of a-subunit • Binds to promoter UP element using s plus a-subunit C-terminal domain • Very strong interaction between polymerase and promoter produces high level of transcription Fig. 26 CTD of a subunit

27. DNase footprint shows a subunit of RNAP can bind UP element • RNAP binds to promoter with an UP element using s plus a-subunit C-terminal domain • End-labeled template (a) or nontemplate (b) rrnBpromoter plus RNAP protein. • Add DNase; if protein bound, DNase does not cut (footprint) Fig. 6.25

28. 6.4 Elongation • After initiation, core RNAP elongates RNA • Nucleotides added sequentially, one after another in process of elongation • Nucleotides enter as triphosphates, but only • a-phosphate enters phosphodiester bond (Fig. 2.9; 3.13) Fig. 3.14

29. Function of Core RNA Polymerase • Core polymerase contains RNA synthesizing machinery • Phosphodiester bond formation involves b- and b’-subunits • These subunits also participate in DNA binding • Assembly of core RNAP is major role of a-subunit

30. Functions of RNAP subunits Purify subunits – urea denatured, then renatured Wild-type and drug-resistant – (Rifampicin blocks initiation) Mix in different combinations Rif-r comes from b subunit Fig. 6.27

31. Role of b in Phosphodiester Bond Formation • Core subunit b lies near active site of RNAP: (affinity-label RNAP with ATP analog, then add [32P]UTP and use SDS-PAGE to see which protein subunits are labeled; Figs. 29, 30) • Active site is where phosphodiester bonds are formed, linking nucleotides • The s-factor may be near nucleotide-binding site during initiation phase Fig. 29

32. Role of b’ and b in DNA Binding Nudler lab showed bothb- and b’-subunits involved in DNA binding: template transfer experiments Two DNA binding sites : Relatively weak upstream site: DNA melting occurs Electrostatic forces predominant Strong, downstream site: hydrophobic forces bind DNA and protein Fig. 32 DNA binding sites for RNAP

33. Structure of Elongation Complex • How do structural studies compare with functional studies of core polymerase subunits? • How does RNAP deal with problems of unwinding and rewinding templates? • How does it move along helical template without twisting RNA product around template?

34. RNA-DNA Hybrids in elongation • Nudler used RNA-DNA crosslinks (Fig. 34) to measure size of hybrid; special reagent in RNA • Area of RNA-DNA hybridization within E. coli elongation complex extends from position –1 to –8 or –9 relative to 3’ end of nascent RNA • In T7 RNAP, similar hybrid appears 8 bp long

35. Structure of T.aquaticus RNAP core (Fig. 35) • X-ray crystallography reveals enzyme shaped like a crab claw: • appears designed to grasp the DNA • Channel in RNAP includes catalytic center • Mg2+ ion coordinated by 3 Asp residues • Rifampicin-binding site • Rif is antibiotic that permits initiation, not elongation

36. Structure of Holoenzyme • Crystal structure of T. aquaticus RNAP holoenzyme shows extensive interface between s and the b- and b’-subunits of core • Predicts s region 1.1 helps open main channel of enzyme to admit dsDNA template to form RPc • After open channel, s expelled from main channel as channel narrows around melted DNA of the RPo • Linker joining s regions 3-4 lies in RNA exit channel • As transcripts grow, have strong competition from s3-s4 linker for exit channel -> often abortive transcripts

37. Structure of Holoenzyme-DNA Complex Crystal structure of T. aquaticus RNAP in synthetic RPo complex • DNA bound mainly to s-subunit • Interactions between amino acids in region 2.4 of s and -10 box of promoter • 3 highly conserved aromatic amino acids participate in promoter melting • 2 invariant basic amino acids in s predicted to function in DNA binding are so positioned • A form of RNAP that has 2 Mg2+ ions Fig. 40

38. Holoenzyme-DNA complex Fig. 41; RNAP bound to special template resembles RPo form

39. Topology of Elongation • Elongation involves polymerization of nucleotides as RNAP travels along template DNA • RNAP maintains short melted region of template • DNA must unwind ahead of advancing RNAP and close up behind it • Strain introduced into template DNA is relaxed by topoisomerases Fig. 44 hypotheses for RNAP movement

40. 6.5 Termination of Transcription • When RNAP reaches terminator at end of gene, it falls off template and releases RNA • 2 main types of terminators: • Intrinsic terminators function with RNAP alone without help from other proteins • Inverted repeat leads transcript to hairpin structure • T-rich region in nontemplate strand produces string of weak rU-dA base pairs holding transcript to template • Other type depends on auxiliary factor called Rho (r): these are r-dependent terminators

41. Inverted Repeats and Hairpins • The repeat is symmetrical around its center shown with a dot • Transcript of sequence is self-complementary • Bases can pair to form a hairpin (lower panel) 5’

42. Structure of an Intrinsic Terminator • Attenuator in trp operon contains DNA sequence that causes premature termination of transcription • E. coli trp attenuator showed: • Inverted repeat allows hairpin to form at transcript end • String of T’s in nontemplate strand result in weak rU-dA base pairs holding transcript to template strand

43. Model of Intrinsic Termination Bacterial terminators : • Base-pairing of something to transcript destabilizes RNA-DNA hybrid • Causes hairpin to form • Hairpin causes transcription to pause • T-rich region nontemplate: • String of U’s incorporated just downstream of hairpin

44. Rho-Dependent Termination • Rho protein caused decreased ability of RNAP to transcribe phage DNAs in vitro • Decrease due to termination of transcription • After termination, RNAP must reinitiate to continue • Rho Affects Chain Elongation (Fig. 48) • Rho Causes Production of Shorter Transcripts (Fig. 49) • Rho Releases Transcripts from the DNA Template (Fig. 50)

45. Mechanism of Rho • No string of T’s in r-dependent terminator, just inverted repeat to hairpin • Rho loads at upstream sequence • Binds to growing transcript, r follows RNAP • Rho catches RNAP as it pauses at hairpin • Rho releases transcript from DNA-RNAP complex by unwinding RNA-DNA hybrid Fig. 51

46. Review questions 6. Diagram difference between a closed and open promoter complex. 9. Diagram four-step transcription initiation process in E. coli 23. Describe expt to determine which subunit is responsible for rifampicin and streptolydigin resistance or sensitivity. AQ. An E. coli promoter recognized by RNAP has -10 box in nontemplate strand: 5’-CATAGT-3’. • Would C-> T mutation at first position be up or down mutation? • Would T-> mutation in last position be up or down?