The process of copying information contained in the DNA of an organism into RNA by RNA polymerase is a crucial step in the biological expression of this information. Consequently, this process is an important junction at which control of gene expression can be exerted. I propose to study a well-characterized system, the interaction of Escherichia coli RNA polymerase with one of the sites on DNA (promoters) at which it can initiate specific RNA synthesis: the PRM promoter of phage lambda, and the control of the process by the cI protein of the phage. I will focus on two regions of DNA, separating them. I wish to probe whether particular positions in each of the two regions are contacted independently of contacts at other positions, and search for determinants in these regions that make a particular step in the pathway to functional complex formation rate limiting. As the spacer DNA is not contacted directly by RNA polymerase, any effects of spacer sequence on promoter function must be transmitted through a particular structure the sequence specifics. By searching for spacer sequences that affect promoter function, we can learn not only about the way E. coli promoters function, but also about DNA sequences that might have altered and interesting structural properties of potential significance in modulating protein-DNA interactions. As model systems for studying the regulation of functional complex formation, I will study the activation of transcription from the PRM promoter by cI protein, and the sensitivity of this and other phage lambda promoters to supercoiling of the DNA. The sequence determinants that render a promoter sensitive to the above two conditions will be examined. In addition, I want to investigate whether there is a correlation (positive or negative) between sensitivity to the two effectors of transcription, as might be expected if control of open complex formation was exerted at the rate limiting step. These studies will be carried out using promoters with particular sequence alterations constructed in vitro, then cloned. Promoters will be characterized in vitro by measuring the rate of functional complex formation, in vivo by assaying a gene product driven by the promoter. With this work I hope to lay a solid foundation for further studies on the regulation of gene expression, in prokaryotes as well as eukaryotes.
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