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

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

5. 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 Bacterial Transcription

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

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

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

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

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

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

13. β β'β' α α ω σ 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 E.coli RNA polymerase Darst et al., 1989

14. β β'β' α α ω σ E.coli RNAP - Sigma Factor 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 σ 70 is the basal sigma factor in E.coli σ 32 is used after heat-shock

15. DNARNAPROTEIN DNA ➙ RNA ➙ PROTEIN transcriptiontranslation Bacterial Transcription 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 Fig 14.6, Genes V (Lewin) Binding Initiation elongation Termination Release

18. Terms 1. TRANSCRIPTION: synthesis of RNA using a DNA template 2.CODING STRAND: the DNA strand that is copied by RNA 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. Fig 14.14, Genes V (Lewin) Typical Bacterial Promoter Sequence Three main parts, the -35, -10 consensus sequences, and the start point. Promoter for σ 70 sigma factor of E.coli

21. PROMOTER - RNAP one 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 Fig 14.6, Genes V (Lewin) Binding Initiation elongation Termination Release

24. RNA being synthesised

25. Bubble RNA pol activities

26. From www.ergito.com website Diagram

27. From www.ergito.com website Model

28. Yeast RNA pol Model

29. Fig 14.11, Genes V (Lewin) 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.

30. Fig 14.12, Genes V (Lewin) 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

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

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

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. Fig 14.15, Genes V (Lewin) Footprint Analysis

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

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 C-rich, G-poor region in RNA preceding termination Fig 16.4, Genes V (Lewin) Rho protein binds to RNA

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

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

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)