10 - Attenuation and sRNA Flashcards
What does hairpin position in mRNA sequence determine
- Transcription termination (Rho
terminator) - Binding site for regulatory factors
- mRNA half life
- Structure of small RNA (sRNA)
- Translation (5’ UTR)
Model for transcription termination
- RNA transcript is held weakly to
template strand by string of weak U-A base pairs - Hairpin begins to form due to the
inverted repeat and destabilises
RNA-DNA hybrid
What are the two elements that terminators consist of
An inverted repeat followed immediately by a A rich regoin in non template strain of the gene
Rho dependent transcriptional terminators
- Hairpin loops occurring at the end of mRNA transcripts
- Rho terminators bind the Rho protein which interacts with the RNApol to stop transcription
Molecular events during termination in bacteria
- Rho forms an open hexamer (6 subunits) on the hairpin
- Rho complex binds to short pyrimidine-rich sequence next to the hairpin
- RNApol contains NusG. NusG interacts with the Rho hexamer which clicks shut to become catalytically active
- Consumption of ATP changes the physical structure of the complex which releases
RNApol from the mRNA
Example of attenuation
- Control of the tryptophan biosynthesis operon
- This is a “cis-acting” mechanism as it concerns the structure of the mRNA that affects whether transcription extension occurs
Example of sRNA
- Control of the expression of RpoS
- This is a “trans-acting” mechanism as it relies on two other elements (sRNA and Hfq) to form a complex that affects the initiation of translation on mRNA
Attenuation
- A mechanism of transcription control that involves premature transcription termination
- Works by terminating transcription before the RNA polymerase reaches the first structural gene of the operon
Example of attenuation
trp operon of E. coli
Tryptophan operon
- Operon responsible for manufacturing the AA tryptophan
- Five genes encode the enzymes needed to make tryptophan (trpABCDE).
- The operator region (trpO) is within the promoter sequence, trpP
- The genes are transcribed as a polycistronic mRNA message of 7 kb which includes the five genes (E,D,C,B,A), the leader sequence (L) and the attenuator region (A).
- The operon is only transcribed when Trp levels are low in the bacterial cell.
- TrpR is a one component regulator and its ligand is tryptophan
Regulation of Trp operon
- Genes are transcribed in the absence of external Trp amino acid
- Genes are not transcribed in the presence of Trp amino acid
How does tryptophan block transcription
The co-repressor tryptophan binds to the aporepressor TrpR dimer, changing the conformation of the DNA binding domain to correct conformation to bind to operator and block transcription
Why is another control mechanism of trp operon needed
- Repression of trp operon is weak, combination of repression and attenuation increases control of repression
- Valuable to cell as trp synthesis requires a lot of energy
Mechanism of attenuation
- Two sites, the trp leader and the trp attenuator, in between the operator and the first gene trpE
- The attenuator contains a transcription stop signal
- When trp is abundant, the attenuator functions and transcription is likely to terminate at the stop signal
- When trp is scarce, the trp operon must be activated so attenuation must be overridden
Role of stem loops in the attenuation sequence at high trp
- Ribosome translates through trp codons and encounters translation stop codon
- Ribosome stops, covering mRNA regions 1 & 2. Polymerase continues to transcribe regions 3 & 4 and the 3:4 termination loop forms
- 3:4 loop binds RNA polymerase and causes its release before reaching trpE
