bacterial transcription dr mike dyall-smith, lab 3.07 aim: understand the general process of...
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Bacterial Transcription
Dr Mike Dyall-Smith, lab 3.07
Aim:
Understand the general process of bacterial transcription
References: Schaecter et al, Microbes, p141-8
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Bacterial TranscriptionMain 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
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Main Points:
a) Overall scheme of information processing in cell
DNA → RNA → Protein (‘central dogma’)
Oscar Miller
Bacterial Transcription
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DNA → RNA → Protein (‘central dogma’)
Bacterial Transcription
In prokaryotes, transcription and translation are directly connected
DNA
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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
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Analysing transcriptsAnalysing transcriptsby Northern Blot hybridisation by Northern Blot hybridisation
Viral transcripts (RNA) separated by agarose gel electrophoresis.
Time post infection
Size of RNA
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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 transcriptsAnalysing transcriptsby Northern Blot hybridisation by Northern Blot hybridisation
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Bacterial TranscriptionRNA 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
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Fig 14.6, Genes V (Lewin)
Phosphodiester bond formation
3’ end
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Bacterial Transcription
• Note deoxythymidine in DNA is replaced by uridine in RNA
QuickTime™ and aTIFF (Uncompressed) decompressor
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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
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β
β'
ααω
σ
E.coli RNA polymerase
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.
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β
β'
ααω
σ
α - 36.5 kDa, enzyme assembly, interacts with regulatory proteins, polymerisation
β' - 155 kDa, binds to DNA templateβ - 151 kDa, RNA polymerisation; chain initiation and
elongationσ - 70 kDa, promoter recognitionω - 11kDa, enzyme stability - restores denatured enzyme
E.coli RNA polymerase
Darst et al., 1989
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β
β'
ααω
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 controlσ70 is the basal sigma factor in E.coliσ32 is used after heat-shock
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DNADNA ➙ RNARNA ➙ PROTEINPROTEINtranscription translation
Bacterial Transcription
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• 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)
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A Transcription Unit
DNA sequence transcribed into an RNA,
from promoter to terminator
Fig 14.6, Genes V (Lewin)
Binding
Initiation
elongation
Termination
Release
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QuickTime™ and aTIFF (LZW) decompressor
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Terms1. 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.
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Terms1. 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
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Fig 14.14, Genes V (Lewin)
Typical Bacterial Typical Bacterial PromoterPromoter Sequence Sequence
Three main parts, the -35, -10 consensus sequences, and the start point.
Promoter for σ70 sigma factor of E.coli
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PROMOTER - RNAPPROMOTER - RNAPone face of the DNA contacts the polymeraseone face of the DNA contacts the polymerase
Fig 14.16, Genes V (Lewin)
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E.coliE.coli Sigma Factors Sigma Factors
Fig 14.16, Genes V (Lewin)
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A Transcription Unit
DNA sequence transcribed into an RNA,
from promoter to terminator
Fig 14.6, Genes V (Lewin)
Binding
Initiation
elongation
Termination
Release
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RNA being synthesised
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Bubble
RNA pol activities
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From www.ergito.com website
Diagram
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From www.ergito.com website
Model
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Yeast RNA pol
Model
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Fig 14.11, Genes V (Lewin)
RNA Pol: Core and Holo enzyme are RNA Pol: Core and Holo enzyme are mainly found on DNA.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.
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Fig 14.12, Genes V (Lewin)
RNA Pol: finding promoters quicklyRNA 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
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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 quicklyRNA Pol: finding promoters quickly
?
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Fig 14.8, Genes V (Lewin)
Initial contact -55 to +20 = ~ 75 bp
RNA Pol binding to a promoter
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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)
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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)
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Fig 14.15, Genes V (Lewin)
Footprint Analysis
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Fig 14.15, Genes V (Lewin)
Footprint AnalysisRNAP+ RNAP-
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Transcription Unit
DNA sequence transcribed into an RNA,
from promoter to terminator
Fig 14.14, Genes V (Lewin)
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Transcription termination: Transcription termination: Intrinsic terminatorIntrinsic 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)
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Transcription termination: Transcription termination: Rho dependent terminatorRho dependent terminator
C-rich, G-poor region in RNApreceding termination
Fig 16.4, Genes V (Lewin)
Rho protein binds to RNA
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Commercial transcription systemsCommercial transcription systems
Fig 16.4, Genes V (Lewin)
Phage RNA polymerases (T3, T7, SP6)
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Transcription - the movieTranscription - 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.
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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
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Transcription stages
Fig 14.6, Genes V (Lewin)