Lecture 10 - Attenuation and sRNA Flashcards
Describe the S+F of mRNA secondary structures
- Bpairing of inverted repeats = stem loops
- Transcrip termination, binding for regulation, half-life, translation, Hfq stability
What is the structure of the transcription termination signal
inverted repeat into stem loop + A rich region after repeat
Describe the 3 step process of transcription termination
- RNAP transcribed polycistronic mRNA
- RNAP recog stop signal
- RNAP falls off and mRNA released
What are Rho-dependent terminators?
Rho protein binds loops @ ends of mRNA and interact with RNAP to stop transcription
Describe the 4 step process of Rho dependent termination
- Rho forms open hexamer on hairpin
- Rho hexamer binds A rich region
- RNAP pauses so NusG interacts with Rho = closed active hexamer
- ATP to release RNAP from mRNA
Describe the structure of the trp operon
- 5 coding regions trpA-E with E first = huge so high energy
- regulation = A inside L inside O inside P
What regulates the trp operon?
- trans-acting TrpR 1 component system = derepressed when low trp
- TrpR aporepressor x2 + trp ligand = repressor
Explain the negative regulation of the trp operon by TrpR repressor during high and low trp levels
- TrpR repressor = always low expression = binds trp O depending on trp levels
- low trp = inactive TrpR = transcription
- high/environ trp = active TrpR = blocks O
Why is attenuation still needed for negative regulation of the trp operon
- TrpR always expressed but low affinity for O = 70x decrease
- Attenuation = 10x decrease so 700x total
- trp operon high energy
What is attenuation and how does it affect negative regulation of the trp operon in high and low trp levels?
- Transcription control using premature transcription termination before structural genes
- low trp = whole mRNA
- high trp = sRNA from +1 to attenuator site
Describe the structure of the transcription stop signal for attenuation
- inverted repeat = hairpin loops with 8 A-T pairs
- 1:2 loops with short leader peptide -> trp codons and translation stop -> 3:4 loops with U rich attenuator
Explain how attenuation works for the trp operon under high trp levels
- transcrip and translation coupled bc SD ready as soon as RNAP moves
- translation @ leader peptide to stop codon, RNAP makes 3:4 loop with termination = RNAP released before trpE
Explain how attenuation works for the trp operon under low trp levels
Translation begins but no tRNAs bc no trp = early pause @ 1:2 loop = no termination signal bc no 3:4 loop = whole mRNA
What are sRNAs and what is their general function?
- Short RNAs 100-400bp, intergenic and never translated, stem loops
- Rho termination + Hfq protein = regulate transcription initiation
- cis or trans acting
What is Hfq and its function?
- Host factor Q = chaperone - Binds RNA to increase half-life and protect from RNases
How does sRNA and Hfq work together to regulate gene expression?
- 6x Hfq + sRNA = mRNA scanning complex so sRNA binds mRNA
- Sequester SD = no translation
- Open mRNA loop = translation
What is the S+F of RpoS?
- Stationary phase gene expression via stimuli
- mRNA 5’UTR with stem loop blocking SD = no translation
What does derepression of RpoS translation require?
- DsrA = sRNA intergenic space with 3 stem loops, cold shock
- Hfq = binds DsrA to stabilise and protect from RNase E
Explain how the DsrA-Hfq complex derepresses RpoS translation
- Complex binds RpoS 5’UTR = prevents hairpin = SD access
- Specific part of 5’UTR = up/down regulation by freeing/blocking SD
