In order to understand competence gene regulation in H. influenzae
, we have been using the huge body of knowledge surrounding E. coli
CRP to inform us about how the ortholog in H. influenzae
functions as a transcription factor. Recently, bandshift analysis of H. influenzea
CRP (HiCRP) binding to DNA has confirmed that both HiCRP and E. coli
CRP (EcCRP) have the same perfect binding site (called ICAP, see an earlier blog about ICAP
). However, I also discovered that HiCRP is much more selective about which sites it will bind; for example, HiCRP will not bind to E. coli
’s lacZYA promoter, even though EcCRP has very high affinity for this site.
Eleven amino acids in EcCRP’s helix-turn-helix DNA-binding domain make direct contact with bases and/or phosphates in the ICAP DNA site; EcCRP makes fewer direct contacts with the lacZYA CRP site because some of the base pairs in this site are not optimal. HiCRP’s highly selective binding behaviour suggests that it needs to make a greater number of direct contacts with the DNA to manifest a binding strength that EcCRP can accomplish with fewer interactions. In other words, more amino acids in HiCRP have to make specific base interactions and/or non-specific phosphate interactions with DNA for the protein to be able to hold on.
I have already identified a single mutation (lacZ-19A) that converts the lacZYA site into something that HiCRP can bind. However, HiCRP still binds to ICAP with >100-fold greater affinity than binding to lacZ-19A. Now it becomes a simple question of which bases in lacZ-19A prevent perfect binding by HiCRP? Six out of twenty-two bases differ between ICAP and lacZ-19A. Three of these differences are likely irrelevant or make minor contributions to binding, so I will ignore them at first. Of the remaining three, it is possible that no change alone will have a dramatic difference but that a particular combination of two key bases will have a dramatic effect. Thus, multiple possible permutations of mutated lacZ-19A sites arise, and it will be a lot of work to test all of them (so I won’t).
So, the simplest experiment is to make 3 single point mutations, but do I change ICAP to resemble lacZ-19A and measure the deleterious effects on binding, or do I change lacZ-19A to resemble ICAP and measure the positive effects? Because there is such a dramatic difference in HiCRP affinity for these two sites, I have good power to detect beneficial or deleterious changes. I am inclined to work with ICAP because I can measure the effect of each mutation while all other bases remain “perfect”. With lacZ-19A, I don’t know if the mutations I introduce would have greater or lesser effects on affinity if studied in the context of other naturally occurring CRP sites. The key question remains “which bases in a CRP site are most critical for HiCRP binding?” and not “why is the lacZYA site such a crap site for HiCRP”. Therefore, I will work with ICAP, and hopefully this will provide insight into the lacZYA issue.