#11 ch19.ppt
TRANSCRIPT
Chapter 19
Prokaryotic Transcription
19.1 Introduction
• RNA synthesis (transcription) occurs in 5′ - 3′ direction on a template strand (antisense strand) that is 3′ to 5′.
• Coding strand (sense strand) – the DNA strand that has the same sequence as the mRNA.
• RNA polymerase – an enzyme that synthesizes RNA using a DNA template (a DNA-dependent RNA polymerase).
• Primary transcript: initial product of transcription, original unmodified RNA product
5’ 5’
5’ 5’ 3’
3’ 3’
3’
Gene A Gene B
Gene A Gene B 5’ 5’ 3’
3’ 5’
5’ 3’
3’
19.1 Introduction
• RNA synthesis (transcription) occurs in 5′ - 3′ direction on a template strand (antisense strand) that is 3′ to 5′.
• Coding strand (sense strand) – the DNA strand that has the same sequence as the mRNA.
• RNA polymerase – an enzyme that synthesizes RNA using a DNA template (a DNA-dependent RNA polymerase).
• Primary transcript: initial product of transcription, original unmodified RNA product
5’ 5’
5’ 5’ 3’
3’ 3’
3’
Gene A Gene B
Gene A Gene B 5’ 5’ 3’
3’ 5’
5’ 3’
3’
19.1 Introduction
• promoter – A region of DNA where RNA polymerase binds to initiate transcription.
• terminator – A sequence of DNA that causes RNA polymerase to terminate transcription.
• transcription unit – A DNA sequence from promoter to terminator; it may include more than one gene (= poly-cistronic).
• startpoint – The position on DNA corresponding to the first base incorporated into RNA. It is given the value of +1; immediate upstream of +1 is -1 (no 0).
19.1 Introduction
Figure 19.02
• Downstream: after the start site (+1). • Upstream: before the start site (begins with -1).
19.2 Transcription Occurs by Base
Pairing in a “Bubble” of Unpaired DNA
• The length of the bubble is ~12 to 14 bp, and the length of RNA-DNA hybrid within it is ~8 to 9 bp.
• Transcription rate: 40 – 50 nt/sec
• Translation rate: 15 aa/sec
• DNA replication rate: ~800 bp/sec
Figure 19.03
19.3 The Transcription Reaction Has Three Stages
• initiation: template recognition and open complex (“bubble”)formation; typically short RNA (<10 nt) is released (abortive transcription) until elongation starts. RNA polymerase does not move.
• elongation: the transcription bubble moves along DNA and the RNA chain is extended in the 5′ to 3′ direction.
• termination: RNA polymerase dissociates and RNA is released.
Figure 19.06
19.4 Bacterial RNA Polymerase Consists of the Core Enzyme and Sigma Factor
• Only one kind of RNA polymerase in E. coli (~13,000 molecules per E. coli).
• Bacterial RNA polymerase holoenzyme can be divided into an core enzyme (α2ββ′ω) that catalyzes transcription and a sigma (σ) subunit that is required only for initiation.
• Sigma factor changes the DNA-binding properties of RNA polymerase so that its affinity for general DNA is reduced and its affinity for promoters is increased = sigma factor is required for promoter binding of RNA polymerase.
• Sigma factor does not bind promoters by itself.
19.4 Bacterial RNA Polymerase
Consists of the Core Enzyme and Sigma
Factor
Figure 19.07
• Half-life of core enzyme – any DNA complex is ~1 hour and sigma factor reduces it to <1 second.
• Sigma factor is responsible for stable interaction of holoenzyme and promoter (half-life of several hours).
19.5 How Does RNA Polymerase Find Promoter Sequences?
Figure 19.08
19.6 Sigma Factor Controls Binding to Promoters
• RNA polymerase binds to the promoter as a closed complex in which the DNA remains double stranded.
• RNA polymerase then separates the DNA strands to form an open complex.
• RNA polymerase incorporates the first two nucleotides and form ternary complex (RNA polymerase + DNA + RNA), which can grow up to ~9 nucleotides-long RNA without movement of RNA polymerase.
• There may be a cycle of abortive initiations before the enzyme moves out of promoter.
• Promoter clearance: RNA polymerase leaves promoter and start elongation.
• Sigma factor may be released from RNA polymerase core enzyme when the nascent RNA is elongated.
• Alternatively, its association may be modified in a way to allow promoter clearance.
Figure 19.10
19.6 Sigma Factor Controls Binding to Promoters
19.6 Sigma Factor Controls Binding to Promoters
• A change in association between sigma factor and core enzyme changes binding affinity for DNA, so that core enzyme can move along DNA.
• Sigma factor is not required for elongation.
Figure 19.11
19.7 Promoter Recognition Depends on Consensus Sequences
• conserved sequence – sequences in which many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations.
• consensus sequences – sequences that represent nucleotides or amino acids most often present at a particular position.
19.7 Promoter Recognition Depends on Consensus Sequences
• The promoter consensus sequences consist of a purine at the startpoint, -10 element (TATAAT; Pribnow box), and another hexamer centered at –35 (-35 element; -35 box).
• Individual promoters usually differ from the consensus at one or more positions.
• Promoter efficiency can be affected by additional elements as well; space between -10 and -35 elements, -35 element upstream (e.g., UP element).
19.7 Promoter Recognition Depends on Consensus Sequences
Figure 19.12
Sigma factor RNA polymerase α subunit
19.8 Promoter Efficiencies Can Be Increased or Decreased by Mutation
• Down mutations that decrease promoter efficiency usually decrease conformance to the consensus sequences, whereas up mutations have the opposite effect.
• Mutations in the –35 sequence can affect initial binding of RNA polymerase (i.e., closed complex formation). However, they do not affect the rate of open complex formation.
• Mutations in the –10 sequence affect formation of closed complex or open complex, or both.
• -10 and -35 element interact with sigma factor. • -10 element is crucial for “melting”
19.9 Multiple Regions in RNA Polymerase Directly Contact Promoter DNA
• σ70 changes its structure to expose its DNA-binding regions when it associates with core enzyme.
• N-terminal region masks DNA-binding domains (DBDs); however, DBDs are exposed upon binding to core enzyme.
Figure 19.15
19.11 Bacterial Transcription Termination
• Terminator (t): DNA sequence that ends transcription.
• Actual signal for transcription termination often lies in RNA.
• Most common signal is a hairpin structure in the RNA product. Figure 19.19
19.12 Intrinsic Termination Requires a
Hairpin and U-Rich Region
• Intrinsic terminators do not require auxiliary protein factors.
• They consist of a G-C-rich hairpin in the RNA product followed by a U-rich region in which termination occurs.
Figure 19.20
• U-rich region destabilizes RNA-DNA hybrid when RNA polymerase pauses at the hairpin.
19.13 Rho Factor Is a Site-Specific Terminator Protein
• Rho factor is a terminator protein and hexameric helicase.
• Rho binds to a rut site on a nascent RNA and tracks along the RNA to release it from the RNA-DNA hybrid structure.
• rut – An acronym for rho utilization site, the sequence of RNA that is recognized by the rho termination factor. It is upstream of the site of termination. Common feature is C-rich sequence.
Figure 19.21
19.15 Substitution of Sigma Factors May Control Initiation
• E. coli has seven sigma factors, each of which causes RNA polymerase to initiate at a set of promoters defined by specific –35 and –10 sequences. E. coli responds to environmental changes by activating specific sigma factors.
Figure 19.26
19.16 Antitermination May Be a Regulated Event • Antitermination: inhibition of transcription termination.
Figure 19.28
Antitermination protein
• Antitermination results in readthrough, which can generate transcripts containing more than one gene.
19.17 The Cycle of Bacterial Messenger RNA
• coupled transcription/translation in bacteria, as ribosomes begin translating an mRNA before its synthesis has been completed.
• Multiple ribosomes move along mRNA polysomes.
• Bacterial mRNA is unstable (degraded from 5’ end) and has a half-life of only a few minutes.
• 3’ end is generated when transcription terminates.
Figure 19.30
19.17 The Cycle of Bacterial Messenger RNA • Multiple mRNAs are undergoing synthesis simultaneously. • Each mRNA carries many ribosomes shown as large dots in
the figure.
Figure 19.31
19.17 The Cycle of Bacterial Messenger RNA
• nascent RNA – An RNA chain that is still being synthesized, so that its 3' end is paired with DNA where RNA polymerase is elongating.
• monocistronic mRNA – mRNA that encodes one protein.
• A bacterial mRNA may be polycistronic in having several coding regions that represent different cistrons.
19.17 The Cycle of Bacterial Messenger RNA
• 5′ UTR (untranslated region) – The untranslated sequence upstream from the coding region of an mRNA.
• 3′ UTR – The untranslated sequence downstream from the coding region of an mRNA.
• 5’ UTR and 3’ UTR regulate translation of mRNA.
Figure 19.32