Why is H. influenzae's CRP so feeble?

One discovery from the grad student's work on Sxy and CRP is that the H. influenzae CRP protein binds CRP sites much less strongly (with much lower affinity) than the E. coli CRP protein does. This is a bit surprising. The two proteins have quite similar sequences, and all the amino acid residues expected to directly contact the DNA are identical.

He's going to contact a lab that has done extensive structural analysis of E. coli CRP, to see how difficult it would be to see how well H. influenzae CRP will superimpose on the E. coli structure. One possibility he suggested is that the dimerization domain of H. influenzae CRP could be weak. This would cause the protein to spend less time assembled into the dimers that most readily bind DNA.

Until now we (at least I) had thought that the affinity of CRP for different genes was determined by how well the gene's CRP site matched the protein's requirements for binding. This would have been optimized for each gene by natural selection acting on mutations in its CRP site. But now I'm wondering whether natural selection has also acted differently on E. coli and H. influenzae CRP proteins to fine tune their affinity for all the sites in their respective genomes.

H. influenzae has only about 40% as many genes as E. coli, and about 40% as many CRP sites regulating them. But I can't think of any way that would favour a 100-fold difference in CRP affinity for the same CRP site, which is what the grad student has found.

The assays were done under exactly the same conditions, but this doesn't ensure that the proteins responded identically to these conditions. I wonder if the binding conditions used for these measurements (optimized for E. coli CRP) might be unsuitable for H. influenzae CRP.

What's UP, Sxy?

One of the grad students has been doing experiments to clarify how the transcriptional activator protein Sxy turns on genes that have its recognition sequence, the "CRP-S" site. He's given me a draft of a paper he's writing about this work.

Sxy works by interacting with another transcriptional activator protein called CRP. CRP's job is to bind to DNA at CRP sites and, by bending the DNA, help RNA polymerase start making RNA. CRP-S sites are much harder to bend than normal "CRP-N" sites, and we have been thinking that Sxy acts by helping CRP to bind (if it can't bend the DNA it lets go).

But several pieces of his data tell us that binding isn't enough. A protein from E. coli (we work mostly in another bacterium, Haemophilus influenzae) binds CRP-S sites fairly strongly in the test tube, but when it's in H. influenzae cells it can't turn the CRP-S genes on without Sxy's help. And changing the CRP-S sequence so H. influenzae's CRP can bind it without Sxy in the test tube doesn't enable CRP to turn the gene on in cells without Sxy's help.

So the grad student is suggesting that Sxy may also affect how RNA polymerase interacts with the DNA. RNA polymerase binds to DNA more effectively if its 'tail' makes contact with an AT-rich sequence called the "UP" sequence. Many genes have an UP sequence, and at normal CRP-N sites CRP is thought to help RNA polymerase's tail bind to it.

He's found that the genes with CRP-S promoters have what look like three precisely-spaced UP sequences beside their CRP-S sites, and in his draft paper he proposes that Sxy acts partly by helping RNA polymerase make contact with these sites. We could test this by changing these sequences to not fit the UP consensus, and seeing if this makes the promoter unable to be activated by Sxy.

This work doesn't address the question of whether Sxy does help CRP bend the DNA. But we won't be able to address this until we have conditions where purified CRP and Sxy both interact. Right now we have the CRP but not the Sxy - it's a very difficult protein to work with.