DNA-bound regulatory proteins interact with RNA polymerase (or associated factors) to control gene expression at the transcriptional level. The long-term goal of the proposed research is to better understand how DNA-bound regulators activate and repress transcription in prokaryotes. The relative simplicity of prokaryotic regulatory systems facilitates basic mechanistic studies that can provide a foundation for the study of transcriptional regulation in higher organisms. The interactions of RNA polymerase (RNAP) with a number of natural activators have been extensively studied. The demonstration that heterologous protein domains fused to RNAP can mediate transcriptional activation simply by providing contact surfaces for DNA-bound proteins provides a valuable new tool with which to explore the mechanism of transcriptional activation. The mechanisms by which such protein-protein contacts activate transcription will be examined by experiments performed in vitro and in vivo. Most prokaryotic activators that have so far been examined appear to interact with either the alpha or the sigma subunit of RNAP. A particularly well-characterized activator that interacts with sigma is the bacteriophage lambda cI protein. The role of the sigma subunit in transcriptional activation will be examined, and the hypothesis that lambdacI activates transcription by stabilizing the binding of a domain of sigma to the promoter -35 region will be tested. A well-established mechanism for transcriptional repression in prokaryotes is simple competitive binding with RNAP. However, recent evidence suggests that some repressors can exert their effects through contact with RNAP. The hypothesis that contact between a DNA-bound protein and RNAP can suffice to repress as well as activate transcription will be tested.
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