The other day we sat down together to take our first joint look at the grant proposal I'm writing. The proposal is still rather inchoate (first time I've used THAT word), especially the experiments section, but it's coming along.
Our model for how the secondary structure of sxy mRNA regulates its expression proposes that the speed of progress of RNA polymerase along the gene determines whether the secondary structure forms before ribosomes can bind and start translating. So I was proposing to directly test whether RNA polymerase pauses or stalls, especially when nucleotides are limited. But one of the grad students pointed out that we first need to show that the secondary structure does indeed block translation. Now she's given me information about a kit that should let us do just that, using cloned wildtype and mutant sxy sequences she's already made.
The kit uses the E. coli translation machinery to translate mRNAs that it makes from a user-provided DNA template with a T7 polymerase promoter. Her sxy clones have this T7 promoter - she's used it to synthesize the RNAs she's used for the RNase digestion analyses. We can use the sxy-1 and sxy-7 mutant RNAs to see if minor changes to the secondary structure change the ability of the E. coli ribosomes to start translation, and we can create a version that lacks most or all of the secondary structure as a positive control, to show how much translation happens in the absence of secondary structure.
It would be scientifically better to do this analysis with H. influenzae translation machinery (rather than E. coli), but we'd need to develop the system from scratch rather than using a kit, and I don't want us to invest that much work into this question. Once we know what happens with the E. coli kit, we can maybe try it with a H. influenzae cell-free lysate.
End of summer &
13 hours ago in The Phytophactor