Bacterial Transcription

1. Bacterial Transcription Dr Mike Dyall-Smith, lab 3.07 Aim: • Understand the general process of bacterial transcription • References: Schaecter et al, Microbes, p141-8

2. Bacterial Transcription Main topics: a) Overall scheme of information processing in cell • DNA ➔ RNA ➔ Protein (‘central dogma’) • Transcription and Translation b) Components of the transcription system in bacteria • RNA polymerase • DNA template, nucleotides, addition of new bases c) Stages of the transcription process • RNAP binding to promoter, DNA unwinding, Initiation, elongation, termination • Consensus promoters, Terminators

3. Bacterial Transcription • Main Points: • a) Overall scheme of information processing in cell • DNA → RNA → Protein (‘central dogma’) Oscar Miller

4. Bacterial Transcription • DNA → RNA → Protein (‘central dogma’) DNA In prokaryotes, transcription and translation are directly connected

5. Bacterial Transcription • Transcription is the synthesis of an RNA molecule, called a transcript, from a DNA template. • Bacteria have only one RNA-P (eukarya have 3) • The bacterial RNA-P enzyme synthesises all the RNA species in the cell • Stable RNAs are tRNA, rRNA • Unstable RNA is mRNA, < 1min 1/2-life

6. Analysing transcriptsby Northern Blot hybridisation Viral transcripts (RNA) separated by agarose gel electrophoresis. Size of RNA Time post infection

7. Analysing transcriptsby Northern Blot hybridisation Viral transcripts (RNA) separated by agarose gel electrophoresis. Size of RNA Time post infection RNA transferred to a membrane, hybridised to a labeled DNA probe to detect viral transcripts

8. Bacterial Transcription RNA polymerase (RNA-P): • Links ribonucleoside triphosphates (ATP, GTP, CTP and UTP) in 5’ - 3’ direction • Copies the DNA coding strand using the template strand • Can be modified to selectively transcribe genes by associating with sigma factors

9. Phosphodiester bond formation 3’ end Fig 14.6, Genes V (Lewin)

10. Bacterial Transcription • Note deoxythymidine in DNA is replaced by uridine in RNA

11. E.coli RNA polymerase RNA polymerase on virus promoters 3D structure (from EM) Oscar Miller E.coli RNAP, ~100 x 100 x 160 Å Darst et al., 1989

12. σ β' α ω α β E.coli RNA polymerase omega factor - function unknown until recently. Darst et al., 1989 sigma factor Core enzyme - will bind to any DNA at low affinity. Selective binding requires the activity of sigma factor.

13. σ β' α ω α β E.coli RNA polymerase α - 36.5 kDa, enzyme assembly, interacts with regulatory proteins, polymerisation β' - 155 kDa, binds to DNA template β - 151 kDa, RNA polymerisation; chain initiation and elongation σ - 70 kDa, promoter recognition ω - 11kDa, enzyme stability - restores denatured enzyme Darst et al., 1989

14. E.coli RNAP - Sigma Factor β' σ α ω α Sigma factor - • allows RNA pol to recognize promoters • reduces affinity to non-specific sites. Specific for particular promoter sequences Several different sigma factors for global control σ70 is the basal sigma factor in E.coli σ32 is used after heat-shock β

15. Bacterial Transcription DNA ➙ RNA ➙ PROTEIN transcriptiontranslation • Transcription occurs at ~ 40 nucleotides/second at 37｡C (E.coli RNA pol.) • Translation is ~ 15 amino acids/sec • Both are much slower than DNA replication (800 bp/sec)

16. A Transcription Unit DNA sequence transcribed into an RNA, from promoter to terminator Binding Release Initiation Termination elongation Fig 14.6, Genes V (Lewin)

18. Terms 1. TRANSCRIPTION: synthesis of RNA using a DNA template 2. CODING STRAND: the DNA strand that is copied byRNA polymerase 3. TEMPLATE STRAND: the DNA strand used by RNA polymerase as the template. It is complementary to the coding strand, and the transcript. 4. TRANSCRIPT: the product of transcription.

19. Terms 1. PROMOTER: The sequence of DNA needed for RNA polymerase to bind and to initiate transcription. 2. START POINT: First base pair transcribed into RNA 3. UPSTREAM: sequence before the start point 4. DOWNSTREAM: sequence after the start point. 5. TERMINATOR: a DNA sequence that causes RNA pol to terminate transcription

20. Typical Bacterial Promoter Sequence Three main parts, the -35, -10 consensus sequences, and the start point. Promoter for σ70 sigma factor of E.coli Fig 14.14, Genes V (Lewin)

21. PROMOTER - RNAPone face of the DNA contacts the polymerase Fig 14.16, Genes V (Lewin)

22. E.coli Sigma Factors Fig 14.16, Genes V (Lewin)

23. A Transcription Unit DNA sequence transcribed into an RNA, from promoter to terminator Binding Release Initiation Termination elongation Fig 14.6, Genes V (Lewin)

24. RNA being synthesised

25. RNA pol activities Bubble

26. Diagram From www.ergito.com website

27. Model From www.ergito.com website

28. Model Yeast RNA pol

29. RNA Pol: Core and Holo enzyme are mainly found on DNA. 500-1000 cRNAP at loose complexes 500-1000 hRNAP at loose complexes Small % free hRNAP 500-1000 hRNAP in closed (or open) complexes at promoters ~ 2500 cRNAP actively transcribing genes. Fig 14.11, Genes V (Lewin)

30. RNA Pol: finding promoters quickly 3 models 1. Random diffusion to target 2. Random diffusion to any DNA, followed by random displacement to any DNA 3. Sliding along DNA Fig 14.12, Genes V (Lewin)

31. RNA Pol: finding promoters quickly 3 models 1. Random diffusion to target 2. Random diffusion to any DNA, followed by random displacement to any DNA 3. Sliding along DNA Too slow Favoured ? Unknown Fig 14.12, Genes V (Lewin)

32. Initial contact -55 to +20 = ~ 75 bp RNA Pol binding to a promoter Fig 14.8, Genes V (Lewin)

33. Transcription occurs inside a region of opened DNA, a ‘bubble’. The DNA duplex is unwound ahead of transcription, and reforms afterwards, displacing the RNA Fig 14.3, Genes V (Lewin)

34. RNA Pol covers less DNA as it progresses from initiation to elongation. Partly because of sigma factor release, and partly from conformational changes of the core enzyme itself. Fig 14.9, Genes V (Lewin)

35. Footprint Analysis Fig 14.15, Genes V (Lewin)

36. Footprint Analysis RNAP+ RNAP- Fig 14.15, Genes V (Lewin)

37. Transcription Unit DNA sequence transcribed into an RNA, from promoter to terminator Fig 14.14, Genes V (Lewin)

38. Transcription termination: Intrinsic terminator • • stem-loop structures, 7-20 bp. • • GC-rich region followed by a poly-U region • Structure forms within transcription bubble, making RNA-P pause • A-U base pairs easily broken, leading to release of transcript Fig 16.3, Genes V (Lewin)

39. Transcription termination: Rho dependent terminator Rho protein binds to RNA C-rich, G-poor region in RNA preceding termination Fig 16.4, Genes V (Lewin)

41. Commercial transcription systems Phage RNA polymerases (T3, T7, SP6) Fig 16.4, Genes V (Lewin)

42. Transcription - the movie DNA (10kb) is attached at one end to a plastic bead, and tethered to a glass capillary. RNAP attached to a plastic bead. Watching single RNA polymerase enzymes move along a DNA template

43. Summary of Bacterial Transcription • Know the main terms in this process • Understand the process: • Template recognition: RNAP binds dsDNA • DNA unwinding at promoter • Initiation (short chains, 2-9nt, made) • Elongation (RNA made) • Termination (RNAP and RNA released) Next lecture on regulation of gene expression

46. Transcription stages Fig 14.6, Genes V (Lewin)