Monday, July 30, 2007

Excited about UP elements

Now that our "Regulation of Sxy" manuscript is submitted, I can shift gear and get back to working on the A/T rich sequences in CRP-S promoters that I hypothesize work as UP elements. First some background about UP elements: bacterial RNA polymerases make two specific contacts with promoter DNA, one is the well-characterized recognition of the -10 and -35 sequences by the sigma subunit, and the second is the less-familiar recognition of upstream A/T rich sequences (UP elements) by RNAP's two alpha subunits.

All of H. influenzae's 13 CRP-S promoters have A/T rich sequences resembling UP elements, and I have shown that these sequences are required for transcription of the pilABCD operon. My working hypothesis is that CRP and Sxy act at CRP-S sites to bend DNA and bring the upstream UP elements close to RNAP so that the alpha subunits can make contact with the UP elements. To test this I am going to remove the CRP-S site and some flanking sequence from the promoter, thus bringing the putative UP elements closer to the -10,-35 RNAP binding site. I predict that the pil promoter will then be active in the absence of either CRP or Sxy. In other words, because I have cut up the DNA and positioned the UP elements physically adjacent to the -10,-35 site, CRP and Sxy are no no longer needed to bring the UP elements close to RNAP by bending the intervening DNA.

Wednesday, July 18, 2007

Manuscript writing/polishing

The latest round of experiments using T7 RNA polymerase in an E. coli S30 in vitro transcription/translation system has yielded very satisfying results. The results are so consistent with our model of sxy regulation that I couldn't have faked better data. Now I am integrating these latest results into our highly polished Sxy manuscript. This integration is fairly easy, but doesn't feel very satisfying (so my mind keeps wandering to my next project). Our manuscript has such an abundance of good data that my new results don't tell us anything new, instead they simply reinforce our old model of sxy regulation. Let's hope that the one reviewer that has been holding out for these results will be satisfied.

Thursday, July 12, 2007

Success with T7

During the past few days I have conducted an experiment to test whether cooling T7 RNA polymerase to 25º allows sxy gene transcripts to fold in vitro as they do in vivo. Although I have not directly tested whether sxy transcripts are folding into the structures we have previously determined experimentally, conducting in vitro transcription/translation at 25º yields results much more in line with what we have detected in vivo. The new results are that sxy-1 is translated at high levels, whereas wildtype and sxy-7 transcripts are not. I am currently repeating the experiment and am also allowing the reactions to proceed for up to 4 hrs to see whether a difference emerges between the two poorly-translated transcripts (WT and sxy-7).

Sunday, July 08, 2007

Trying to make T7 RNAP behave like bacterial RNAP

I have been using coupled in vitro transcription/translation to test whether the secondary structure at the 5' end of sxy transcript blocks translation. Our strong genetic and in vivo results suggest that the sxy-1 mutation weakens 2º structure and so facilitates ribosome binding; the sxy-7 mutation does the opposite.

My in vitro results show a very little bit more translation in sxy-1 relative to wildtype, and about half as much translation with sxy-7. This trend is nowhere near as dramatic as the in vivo results, so Rosie and I have been troubleshooting the in vitro assay in an attempt to make the test tube more like in vivo conditions. A central complication is that the the in vitro assay uses the phage T7 RNA polymerase to achieve high levels of transcription. T7 RNAP is a very fast and processive (ie. it never drops the ball) enzyme. T7 RNAP transcribes about 10 times faster than E. coli RNAP, and this high-speed can result in miss-folding of RNA (Lewicki et al. (1993) showed this by using T7 RNAP to transcribe the 23S rRNA gene: the T7 product was non-functional, but melting and renaturation restored 23S functionality). Yesterday, I repeated the in vitro experiments at 25ºC because Lwicki found that this low temperature slowed T7 RNAP to a speed similar to E. coli RNAP.

We were also concerned that using circular templates for transciption might result in poly-cistronic transcripts; in other words, T7 RNAP may continue to transcribe around the plasmid template and never stop, resulting in a potentially miss-folded string of sxy genes. To test whether this is a problem, I linearized some of my templates before transcription/translation. I will have the results by Thursday, and our modified proceedures may yield results more like our in vivo data.

Wednesday, July 04, 2007

Back in the lab

Now that I'm back in the lab, I have two primary goals: 1) Finish the experiements required for our Sxy manuscript, 2) Conduct a few more experiments to beef up our CRP-DNA binding manuscript. For #1, I will again assay the translatability of various sxy gene mutants. This is a replication of an earlier experiment so it should go smoothly. Preliminary results support our model in which strong sxy 2º structure reduces translation; if I confirm these results, the manuscript is done. I may include an experiment to test whether a short DNA oligo can compete with formation of sxy 2º strucutre and thus facilitate translation of sxy mRNA. I attempted this experiment before, but found that the short mRNAs I was using encode a half size Sxy protein that is not detected by our anti-Sxy antibodies. I can now repeat this experiment using my new clones that generate full-length sxy mRNA.
For goal #2, Rosie and I may decide to resubmit the CRP-DNA binding manuscript as is, or I can conduct a few more experiments to improve it. Today I will write a draft of the proposed experiments so that we can weigh our options.