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.
1 day ago in Variety of Life