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
Role of stem loops in the attenuation sequence at low trp
- The TrpR aporepessor does not bind and the level of transcription is very high
- The ribosome binds the leader sequence to translate the peptide but the concentration of tRNA charged with Trp is very low and so
the translation stalls - This means that the 2:3 stem
loop can form instead of the 3:4
loop. - The 2:3 stem has no transcription
Termination signal so RNA
polymerase proceeds to generate a transcript for the entire operon
Other examples of attenuation
Other E. coli operons use the attenuation mechanism eg. histidine operon
Small RNA (sRNA)
- Encoded in intergenic regions between genes in operons
- Never encodes a protein
- Contains stem loops and often forms a Rho termination sequence
Host factor Q
- RNA binding protein
- Chaperone that binds sRNA and protects them from degradation by RNAses
Mode of action of sRNA and Hfq
- Complex of sRNA with Hfq binds matching mRNA sequences
- Results in either sequestering the shine dalgarno site to prevent translation
- Or opens a mRNA stem loop in the mRNA to allow translation
RpoS gene functions
- Glycolysis
- Fermentation
- Anaerobic respiration
- Stress responses to cold, oxidative challenge, starvation
Secondary structure of RpoS mRNA
- mRNA of RpoS has a 5’ untranslated leader sequence
- This forms a stem loop occluding the SD site and inhibiting translation
Two elements required for repression relived
Two elements required DsrA and Hfq
DsrA
- Small RNA encoded in an intergenic region
- Does not encode a protein
- Expressed at high levels at
temps less than 20 ºC
How is translational repression relieved
Hfq/DrsA forms a complex which
binds to the 5’ UTR of RpoS
mRNA, preventing hair pin
formation, promoting translation
of RpoS
How many genes does DsrA regulate
At least 36
How many sRNAs does Hfq bind
atleast 22
Characteristics of sRNAs
- Frequently end in a Rho terminator
- Multiple stem loops
- Often encoded in DNA
intergenic spaces between
protein encoding genes - Polycistronic mRNA transcript is processed to release the sRNA which can be detected by northern blot
Decection of sRNAs
- Immunoprecipitation of Hfq and analysis of bound sRNA
- Detect transcripts using a microarray of all intergenic spaces in a given genome
- Direct cloning of sRNA in a given size range
- Deep sequencing
How can sRNAs influence post transcriptional and translational regulation
- Targeting mRNA for degradation
- De-repression of SD, hence translation
- Occlusion of SD, hence repressing translation
