E. coli sigma-70 belongs to a large family of bacterial sigma factors. Sequence similarity between the family members extends through 4 conserved sequence segments, numbered 1-4, of which regions 2 and 4 are the most highly conserved. Sigma-70 consists of at least two independent folding domains. The first contains conserved region 2 and specifically binds to DNA containing -10 promoter elements, while the second contains region 4 and specifically binds to DNA containing the -35 element. No direct structural data is available for any member of the sigma family; however, region 4 contains a helix-turn-helix (HTH) DNA binding motif, and residues required for -35 recognition are found in the second (recognition) helix of the HTH. My previous work has established that of the HTH proteins of known structure, the cro and cI repressors of bacteriophage 434 are the most similar in sequence to region 4 of the sigma family. This result has been confirmed by others on the basis of structure profiles. I have also extended earlier results from our lab which suggested that amino acids downstream from the region 4 HTH interact with transcription activators, by showing that 4 residues in this region contribute to transcription activation by the CRP protein. I have used the Computer Graphics Lab facilities to examine the implications of my structural hypothesis for sigma-70 and resulting implications of the CRP-transcription activation results. I modeled sigma-70 region 4 based on the 434 repressor DNA co-crystal structure. I then fit this model into the co-crystal structure of CRP-cAMP-DNA, such that the promoter-CRP site spacing was maintained. Interestingly, two of the mutants are at residues which contact DNA in the model, while the other two are oriented such that they can contact the surface of CRP at a site previously identified as important for transcription activation. I plan to test these structural insights using in vitro transcription assays and DNA footprinting. I also plan to refine and test the model further using the CGL facilities.
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