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Transcription in bacteria, Transcription, The operon - Coggle Diagram
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Transcription
E. coli RNA polymerase
- Contains enzyme active site
Sigma (σ) factor
Promoters recognized by different σ factors in E. coli
- σ70 (primary σ factor) = “housekeeping” genes
- σS = stationary phase genes
σ70 promoters
- -35 element (start of transcription = “+1”)
- Spacer (16-18 bp; 17 bp optimal)
-10 region.
Transcription requires specific sequences in template DNA to direct & modify RNA polymerase activity.
- RNA polymerase interacts with PROMOTERS.
- Specific DNA sequences (including -35, -10 motifs) at defined points upstream of transcription start point (+1)
- Regulatory proteins also bind within this region & act to increase or decrease polymerase activity
Key Concept: proteins such as polymerases directly interact with bases in nucleic acid- as DNA- binding proteins they "read"/ recognise DNA sequence.
Sigma factors determine promoters bound by
core RNA pol
- In prokaryotes, RNA polymerase core enzyme cannot directly
bind / recognise promoter sequence
- Separate subunit known as sigma () recognises two sequences
at -10 and -35 (with some variation)
- Different sigma proteins (e.g. σ70, σS) recognise different promoter sequences.
- Examples of sigma 70 promoters from several genes in E. coli -
all “strong” (highly expressed by RNAP)
- Consensus sequence - when you align multiple promoter
sequences & look for most common base at every position - is "optimal" sequence.
70 Promoter Consensus Sequence
- Promoter activity may also be regulated by additional
proteins (transcription factors) however, RNAP binding sequence can regulate levels of expression.
- Similar to consensus usually indicates “strong” promoter.
- f promoter is “weak” then RNA polymerase has lower affinity
- less mRNA produced
Transcription in prokaryotes:
- Core enzyme forms complex with sigma factor
- Complete enzyme forms a
complex with the promoter
- DNA is unwound.
- RNA synthesis begins by
adding complimentary NTP at +1
- Sigma factor dissociates
- RNA synthesis continues
until an appropriate termination signal.
Transcription termination.
- May involve protein factors (e.g. Rho) or may be an intrinsic property of mRNA sequence.
- Most common transcription terminator is hairpin loop formed
- Hairpin loop structure interferes with enzyme active site,
destabilising process & causing RNA molecule, RNAP & template DNA to dissociate.
Error Rates in E.coli Transcription.
- Relatively high - approximately 1 in 104
- RNAP does not proofread (c.f. DNA polymerases)
Regulation of Transcription - Rationale
- Most bacteria have to express the correct phenotype to
survive (& be “fit” in evolutionary terms) across range of environments e.g. E. coli lives in human gut & water / soil.
- Some bacteria have “complex” developmental lifecycles
(e.g. spore formation)
- This requires regulation of gene transcription, so they are
expressed when required (& not otherwise)
- Constitutive (constant) expression would be wasteful
- Gene expression can be regulated at transcription stage or
later (translation)
Regulation of Transcription - Mechanisms 2 basic types of transcriptional regulatory mechanism
- Positive regulation (Activation)
- Negative regulation (Repression)
Both are carried out by transcription factors
- DNA-binding proteins
- interact with promoters
- Modulate gene expression
May affect different stages of transcription initiation –
often promoter binding by RNAP
- Simple negative mechanism – steric hindrance of RNAP binding to promoter DNA
- Simple positive mechanism – improving weak promoter affinity for RNAP
The operon
Bacterial genes are organized into operons: clusters of co- regulated genes involved in same process (e.g. biochem pathway) & transcribed onto single polycistronic mRN
- Eukaryotic genes are normally dispersed “randomly” through genome & each transcribed onto separate monocistonic mRNA
in addition to being physically close (clustered) in genome, genes in an operon are co-regulated so that they all turn on or off together.
- Grouping related genes under common control
mechanism allows bacteria to adapt rapidly to environmental changes.
- Best-studied operons from E. coli - encode enzymes for:
- Lactose utilisation (lac operon)
- Tryptophan biosynthesis (trp operon)