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Bacterial Transcription Dr Mike Dyall-Smith, lab 3.07 Aim: Understand the general process of bacterial transcription Reference: Schaecter et al, Microbes, p141-8 Bacterial Transcription Main Points: 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 Bacterial Transcription DNA ! RNA ! Protein (‘central dogma’) a) Overall scheme of information processing in cell DNA ! RNA ! Protein (‘central dogma’) DNA Oscar Miller Bacterial Transcription In prokaryotes, transcription and translation are directly connected Analysing transcripts by Northern Blot hybridisation Transcription is the synthesis of an RNA molecule, called a transcript, from a DNA template. Bacteria have only one RNA-P (eukarya have 3) Viral transcripts (RNA) separated by agarose gel electrophoresis. Size of RNA 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 Time post infection Analysing transcripts by Northern Blot hybridisation Bacterial Transcription RNA polymerase (RNA-P): Links ribonucleoside triphosphates (ATP, GTP, CTP and UTP) in 5’ - 3’ direction Viral transcripts (RNA) separated by agarose gel electrophoresis. Size of RNA Copies the DNA coding strand using the template strand Can be modified to selectively transcribe genes by associating with sigma factors Time post infection RNA transferred to a membrane, hybridised to a labeled DNA probe to detect viral transcripts Bacterial Transcription Phosphodiester bond formation • Note deoxythymidine in DNA is replaced by uridine in RNA 3’ end Fig 14.6, Genes V (Lewin) E.coli RNA polymerase RNA polymerase on virus promoters E.coli RNA polymerase omega factor function unknown until recently. 3D structure (from EM) Darst et al., 1989 $ !' " # sigma factor " ! Oscar Miller E.coli RNAP, ~100 x 100 x 160 Å Darst et al., 1989 Core enzyme - will bind to any DNA at low affinity. Selective binding requires the activity of sigma factor. E.coli RNA polymerase $ !' !' " # Darst et al., 1989 E.coli RNAP - Sigma Factor " " ! ! ! - 36.5 kDa, enzyme assembly, interacts with regulatory proteins, polymerisation "' - 155 kDa, binds to DNA template " - 151 kDa, RNA polymerisation; chain initiation and elongation # - 70 kDa, promoter recognition $ - 11kDa, enzyme stability - restores denatured enzyme • • • ! Sigma factor • allows RNA pol to recognize promoters • reduces affinity to non-specific sites. Several different sigma factors for global control $70 is the basal sigma factor in E.coli $32 is used after heat-shock A Transcription Unit PROTEIN translation 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) $ Specific for particular promoter sequences Bacterial Transcription DNA ! RNA transcription " # Binding DNA sequence transcribed into an RNA, from promoter to terminator Initiation Release Termination elongation Fig 14.6, Genes V (Lewin) 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. Terms Typical Bacterial Promoter Sequence 1. PROMOTER: The sequence of DNA needed for RNA polymerase to bind and to initiate transcription. Three main parts, the -35, -10 consensus sequences, and the start point. 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 Promoter for $70 sigma factor of E.coli Fig 14.14, Genes V (Lewin) PROMOTER - RNAP one face of the DNA contacts the polymerase Fig 14.16, Genes V (Lewin) E.coli Sigma Factors Fig 14.16, Genes V (Lewin) A Transcription Unit Binding DNA sequence transcribed into an RNA, from promoter to terminator Initiation Release Termination elongation Fig 14.6, Genes V (Lewin) RNA being synthesised RNA pol activities Diagram Bubble From www.ergito.com website Model Model Yeast RNA pol From www.ergito.com website RNA Pol: Core and Holo enzyme are mainly found on DNA. RNA Pol: finding promoters quickly 3 models 1. Random diffusion to target 500-1000 cRNAP at loose complexes 500-1000 hRNAP at loose complexes Small % free hRNAP 2. Random diffusion to any DNA, followed by random displacement to any DNA 500-1000 hRNAP in closed (or open) complexes at promoters ~ 2500 cRNAP actively transcribing genes. 3. Sliding along DNA Fig 14.11, Genes V (Lewin) Fig 14.12, Genes V (Lewin) Initial contact -55 to +20 = ~ 75 bp RNA Pol: finding promoters quickly 3 models 1. Random diffusion to target Too slow 2. Random diffusion to any DNA, followed by random displacement to any DNA Favoured Unknown Fig 14.12, Genes V (Lewin) ? RNA Pol binding to a promoter 3. Sliding along DNA Fig 14.8, Genes V (Lewin) 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) Footprint Analysis 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) Footprint Analysis RNAP+ Fig 14.15, Genes V (Lewin) Fig 14.15, Genes V (Lewin) RNAP- Transcription termination: Intrinsic terminator Transcription Unit • stem-loop structures, 7-20 bp. DNA sequence transcribed into an RNA, from promoter to terminator • GC-rich region followed by a polyU region • Structure forms within transcription bubble, making RNA-P pause • A-U base pairs easily broken, leading to release of transcript Fig 14.14, Genes V (Lewin) Fig 16.3, Genes V (Lewin) 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) Commercial transcription systems Phage RNA polymerases (T3, T7, SP6) Fig 16.4, Genes V (Lewin) 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 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 Transcription stages Fig 14.6, Genes V (Lewin)