The objective of the proposed research is to study the mechanisms of transcriptional activation in prokaryotes. The analysis of relatively simple and well-characterized prokaryotic systems continues to provide insight into gene regulation in higher organisms, contributing to the understanding of abnormal and normal cellular processes such as oncogenesis and embryogenesis. At its simplest, transcriptional activation involves the interaction of a single DNA-bound activator with RNA polymerase, and the proposed research will examine one such interaction in detail. In more complex cases, multiple DNA-bound activators function synergistically. Through the analysis of two new examples of transcriptional synergy in E. coli, we hope to shed light on the basic underlying mechanisms in both prokaryotes and eukaryotes. Recent evidence suggests that the bacteriophage lambda cI protein (lambda cI) activates transcription from the lambdaP PM promoter through an interaction with the sigma subunit of RNA polymerase. Biochemical and genetic confirmation of this proposed interaction will be sought, and the specific amino acids involved in this interaction will be identified by isolating RNA polymerase mutants altered in their responses to wild type and mutant forms of lambda cI. Unlike lambda cI, the E. coli Cyclic AMP Receptor Protein (CRP) activates transcription from a variety of different positions with respect to the startpoint of transcription, in some cases contacting the alpha subunit of RNA polymerase. CRP can also activate transcription synergistically when two CRP-binding sites are appropriately positioned upstream of a CRP- responsive promoter. This synergy is evidently not due to the cooperative DNA binding, an expected mechanism in prokaryotes. Experiments are proposed to test the hypothesis that both DNA-bound CRP molecules interact directly with RNA polymerase and to identify the relevant points of contact on both CRP and RNA polymerase. CRP can also function synergistically with lambda cI when the two activators are bound at an artificial promoter bearing binding sites for both proteins. The hypothesis that synergistic activation by lambda cI and CRP involves their contact with the sigma and alpha subunits of RNA polymerase, respectively, will be tested.
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