One of the grad students and I were talking this afternoon, trying to develop a model of how the regulatory proteins Sxy and CRP interact to cause transcription from promoters with CRP-S sites. We wound up with a model where Sxy does two things that increase transcription at these otherwise-unfavourable promoters.
The first thing would be that Sxy helps CRP bind to and bend DNA right at the CRP-S site. At CRP-N sites, which have an easily-bendable sequence, Sxy isn't needed for CRP binding.
The second thing would be that Sxy also interacts with another attribute of CRP-S promoters, and together with CRP helps RNA polymerase to begin transcription. This 'other attribute' is probably outside of the core CRP-S site; it could be a part of the nearby sequence that we haven't examined yet, or something about the separation of the CRP and RNA polymerase binding sites. If CRP binds to such a site without Sxy, it can't initiate transcription.
The other component of our model is that there is much more CRP than Sxy in the cell, so most of the CRP isn't associated with Sxy, but most of the Sxy is associated with CRP.
This model explains why changing the core of a CRP-S site into a CRP-N type core doesn't allow the promoter to work nearly as effectively as an authentic CRP-N site. And why doing the reverse (changing a CRP-N core into a CRP-S core) also creates a lousy promoter.
In our sketches we always had Sxy associate with CRP in solution, before either made contact with the DNA. But I'm wondering if this is necessary, or if one or the other protein might bind DNA and then its partner. Probably it is necessary. E. coli CRP alone binds very poorly to CRP-S sites in vitro; H. influenzae CRP won't bind them at all. So either Sxy must bind the DNA at CRP-S sites before the CRP gets there, or they must meet up in solution. Sxy has none of the features of typical DNA-binding proteins.
Of course, it could be that Sxy doesn't bind CRP at all, but instead binds to RNA polymerase....
Kurt Gödel's Open World
1 day ago in The Curious Wavefunction