Using bandshifts to study CRP-DNA interactions

Along with cloning new pilABCD promoter constructs for testing the role of putative UP elements in CRP-S promoters (see July 30 post), I am purifying E. coli and H. influenzae CRP proteins to test their in vitro affinities for various gene promoters. I have been conducting similar bandshift experiments off and on throughout my graduate studies and have found some very curious and unexpected results (these unexpected results are undoubtedly part of the reason this work has been sporadic; it's sometimes very hard to know what to conclude and what to do next). In short, I found that E. coli CRP behaves, well, like E. coli CRP, which is all well and good. However, H. influenzae CRP (a highly similar ortholog with the same cellular role) exhibits dramatically different binding behaviour. For example, E. coli CRP has very high affinity and specificity for the lacZYA CRP-binding site, whereas H. influenzae CRP does not treat this site any differently from random DNA sequence. As a first step to figure out why, I have mutated the lacZYA CRP site to better match the CRP-binding site consensus sequence (only a single base pair change makes the lacZYA site nearly "perfect"). If this mutation is sufficient to change this site to a specific H. influenzae CRP-binding site, then we will know that the Hflu protein is highly selective for a specific base pair at position 19 in the 22bp site.