Green 14 - Regulatory Small RNAs

Question 1

Where do regulatory sRNAs operate and what do they affect?

Answer 1

sRNAs operate far away, and are regulated separately, to the transcripts that they regulate (work in trans)
they affect the expression of prokaryotic genes

Question 2

List the 4 different ways in which sRNAs regulate gene expression.

Answer 2

transcription interference
transcription attenuation
blocking of SDS (including RNA thermometers)
directly/indirectly stopping translation (OmpF)
Altering nuclease substrate (RNAIII)

Question 3

Give an example of how sRNAs INTERFERE WITH TRANSCRIPTION.

Answer 3

The production of MccA/B enzymes to breakdown Met>Cys in C. acetobutylicum (Gram +ve)

• to do with T box and S box riboswitches that sense the levels of Cys (loaded on tRNAs) and SAM (to indicate how much Met is present)
• we get production of MccA/B with low Cys and high Met because no clash of RNAPs on both transcripts
• with low C and low M we get a clash from RNAPs, cause them to dissociate and incomplete transcripts (degraded) and cannot produce genes for the metabolism of M>C

Question 4

How can high levels of metabolite alter transcription? (transcription attenuation)

Answer 4

high levels of metabolite, expression of sRNA, stabilises the formation of the terminator loop, transcription stops

Question 5

Give an example where the substrate for a nuclease is altered following binding of a sRNA.

Answer 5

S. aureus - binding of RNAIII
- under high levels of infection (quorum sensing), RNAIII produced to regulate a no. virulence factors (one being Staph Protein A - SPA)
- SPA only needed to establish an infection so needs to be turned off @ later stages
- RNAIII binds directly to the 5’ UTR of SPA mRNA and blocks the binding of ribosomes therefore no translation
OR
- the binding creates a substrate for RNAseIII to come and chop up any dsRNA, the broken RNA is then further digested by more nucleases so TRANSLATION IS COMPLETELY STOPPED (IRREVERSIBLE)

Question 6

Describe an example of when translation is blocked DIRECTLY.

Answer 6

• OmpF = B barrel porin that non-selectively takes up substances from the environment (toxic/nutrients)
• OmpF expression blocking in Gram -ve bacteria
  if toxins present/under conditions of high osmolarity, sTFs activated which give rise to the expression & activation of micF
• micF binds to ompF mRNA to stop its translation by blocking the rbs and start codon
• OmpF expression stopped & cell stops taking up toxins (as well as nutrients)
• over time OmpC expressed which is a smaller porin that allows other substrates to be taken up

Question 7

What types of proteins do RNA thermometers allow to be expressed?

Answer 7

• at higher temps, the breakdown of the secondary structure releases the rbs and allows the translation of heat shock genes such as HEAT SHOCK PROTEINS and other VIRULENCE FACTORS (beneficial when bacteria enter a new host eg Listeria)