Field of Science

Possible work with the new rpoD mutation

I'm up to my ears preparing materials for the new version of Useful Genetics, but we have a great new technician through UBC's Science Co-op program.  She's nearly finished the work preparing the RNA samples for our big RNA-seq project, so it's time to consider what she should work on next.

One possibility is characterizing the newly identified rpoD mutant strain.  This strain (RR753) has a point mutation in HI0533, which encodes the sigma factor that regulates initiation and early elongation of transcription of 'housekeeping' genes, especially during exponential growth.

One analysis we need is BioScreen growth curves of the mutant and a wildtype control, to confirm the preliminary growth curve data suggesting that this mutation causes slightly slower cell growth (lower graph).  I think this slower growth results from a general slowing or minor disruption to normal transcription of many genes, and is not specific to its effects on competence. 

My hypothesis is that the mutation's effect on transcription of sxy mRNA increases competence by increasing sxy translation.   I've long hypothesized that slowing elongation or increasing pausing in the 100 nt segment of sxy mRNA that forms its regulatory secondary structure will promote sxy translation by increasing the ribosome's access to the sxy ribosome-binding site and start codon.  We're not in a position to dive into the molecular analyses of RNA and protein that will probably be needed, but I wonder if there are some genetic or culture-conditions approaches that will shed light on the situation.
  • Is RR753 sensitive to the inhibition of competence by added purines?
  • What's the effect of an hfq deletion in this background?
  • How does this strain respond to added cAMP?
  • How does it respond to the standard competence-inducing MIV treatment?
  • Does the mutation increase competence of a sxy mutant (sxy6) that has an extra-stable secondary structure?
  • Does it further increase log-phase competence of the sxy hypercompetence mutants, which have weakened sxy mRNA secondary structures?
I think the technician is going to be doing a lot of competence time-courses...

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