CRP and Sxy plans
Having completed all but a few final touches on the revised Sxy manuscript, I can now return my focus to the next most pressing piece of science/manuscript: an investigation into the molecular mechanisms by which CRP binds and activates CRP-S promoters. The initial (already submitted to and rejected by a journal) version describes experiments that compare how E. coli and H. influenzae CRP bind to DNA, followed by experiments examining how converting a CRP-S site to a CRP-N-like site influences CRP binding and transcription initiation, then some experiments testing Sxy dependence at CRP-S promoters, and finally experiments designed to test the influence of putative UP elements in CRP-S promoters.
This manuscript began life as a chapter in my thesis designed to pull together several good, but not very cohesive, experiments. New experiments conducted over the past few months have allowed this manuscript to evolve into a more thorough analysis of how CRP binds and activates CRP-S promoters. The constantly evolving nature has paved the way for some very neat experiments, however this means that the manuscript won’t be done for another couple of months yet. On the plus side, it will be a much more cohesive manuscript and the science will be more complete and exact (no more hodgepodge of results).
I now feel that the manuscript should be only about CRP and that experiments testing Sxy’s role in DNA binding and transcription activation should be saved for a Sxy-specific manuscript that combines my work with Sunita’s. I think Sunita’s excellent molecular biology skills are bringing us rapidly towards the ability to directly test how Sxy functions on a molecular level. Furthermore, I have recently acquired some V. cholerae DNA which I hope Sunita can use in her Sxy and CRP cross-species complementation experiments. Because very few regions of the Sxy protein are conserved between H. influenzae, E. coli and V. cholerae, if all three proteins work in all three species, this result will very quickly narrow down which regions of the protein are required for whatever function we discover Sxy to have. She and I will meet tomorrow to discuss our experimental and manuscript plans.