Department of Microbiology & Molecular Genetics
Harvard University Medical School
Building D1, Room 306B
200 Longwood Avenue
Boston, MA 02115
1991 Searle Scholar
We use well-characterized prokaryotic regulatory proteins to study the fundamental mechanisms of transcriptional control. The control of gene expression depends on the interactions of DNA-bound regulatory proteins with RNA polymerase, or associated factors, as well as the protein-protein interactions of the regulators themselves. Many transcriptional regulators must dimerize, forming either homo- or hetero-dimers prior to binding the DNA, and they also participate in weaker interactions that occur once they are appropriately positioned on the DNA.
Our current work concerns the following issues:
Our studies on activation are focused on two transcriptional activators, the bacteriophage lambda cI protein (cI) and the E. coli cyclic AMP receptor protein, CRP. Genetic evidence suggests that cI uses a well-defined activation surface to contact the sigma subunit of RNA polymerase. We are using both genetic and biochemical approaches to probe the nature of this interaction and develop a detail model of the activation process. Recently, we have shown that cI and CRP can function synergistically to activate transcription from an artificial promoter bearing binding sites for both proteins. Our experiments suggest that the two DNA-bound activators interact simultaneously with distinct subunits of RNA polymerase. Transcriptional activation in eukaryotes generally involves the action of many regulators, whose synergistic effects are thought to occur because multiple activators can interact simultaneously with the basal transcription apparatus. Though this mechanism has been widely discussed, the complexity of the eukaryote transcriptional machinery makes it difficult to pin down the relevant interactions. Our current work is directed towards the detailed molecular dissection of prokaryotic examples of transcriptional synergy.
Both cI and CRP bind as dimers to their specific recognition sites, and in the case of cI, pairs of DNA-bound dimers interact resulting in cooperative binding of both adjacent and artificially separated sites. Recently, we have developed a genetic selection strategy the permits the isolation of altered dimerization specificity mutants, and we are using this approach to identify the specificity determinants for the dimerization of these proteins as well as several eukaroyotic transcription factors. We are also using genetic and biochemical strategies to probe the higher order interaction of DNA-bound dimers, focusing on cI and its relatives.
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