Monday, July 30, 2007

Excited about UP elements

Now that our "Regulation of Sxy" manuscript is submitted, I can shift gear and get back to working on the A/T rich sequences in CRP-S promoters that I hypothesize work as UP elements. First some background about UP elements: bacterial RNA polymerases make two specific contacts with promoter DNA, one is the well-characterized recognition of the -10 and -35 sequences by the sigma subunit, and the second is the less-familiar recognition of upstream A/T rich sequences (UP elements) by RNAP's two alpha subunits.

All of H. influenzae's 13 CRP-S promoters have A/T rich sequences resembling UP elements, and I have shown that these sequences are required for transcription of the pilABCD operon. My working hypothesis is that CRP and Sxy act at CRP-S sites to bend DNA and bring the upstream UP elements close to RNAP so that the alpha subunits can make contact with the UP elements. To test this I am going to remove the CRP-S site and some flanking sequence from the promoter, thus bringing the putative UP elements closer to the -10,-35 RNAP binding site. I predict that the pil promoter will then be active in the absence of either CRP or Sxy. In other words, because I have cut up the DNA and positioned the UP elements physically adjacent to the -10,-35 site, CRP and Sxy are no no longer needed to bring the UP elements close to RNAP by bending the intervening DNA.


At 10:52 AM, Blogger Sunita Sinha said...

Very neat! These simple experiments should yield very interesting results!

At 9:24 AM, Blogger LindsayAWilson said...

Do CRP-N (i.e. need CRP only) sites also have these UP elements or are they only observed in CRP-SXY CRP-S sites?

At 9:39 AM, Blogger Some call me Tim said...

Lindsay, some (many?) Hflu CRP-N sites are probably adjacent to UP elements, but it is hard to tell using the same method as was applied to CRP-S sites. I identified the putative UP elements by aligning the 13 CRP-S sites in Hflu and this alignment revealed the evenly-spaced A/T runs that I'm calling UP elements. We and others have identified many Hflu CRP-N sites, and these fall into either class I, II, or III whereas CRP-S sites all appear to be class I sites. This class designation describes where CRP binds in relation to RNAP, and by aligning all CRP-N sites, we are trying to align different promoter architectures and this probably obscures our ability to detect UP elements.


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