The goal of this research is to elucidate the molecular mechanisms involved in regulation of transcription, using as model systems two catabolite sensitive operons of E. coli. A variety of physical and biochemical methods will be applied to study interactions of purified RNA polymerase and catabolite activator protein (CAP) with DNA fragments containing wild type or mutant promoter regions of the lactose and galactose operons. The techniques to be used include gel electrophoresis (for the study of DNA-protein binding and for analysis of transcription products), site-directed mutagenesis, nuclease protection experiments, centrifugation, and fluorescence. The project is specifically aimed at study of three issues of current interest in transcriptional control: i. What are the characteristics and roles of the overlapping RNA polymerase binding sites found at many promoters? ii. What is the function of multiple copies of regulatory proteins which may interact at the promoter region? Two CAP molecules are known to bind to the gal promoter. How do these interact with DNA, with RNA polymerase, and with each other to facilitate initiation of mRNA synthesis? Does CAP work in different ways at different operons? iii. Are there interactions between CAP molecules (or between CAP and RNA polymerase) bound to nonadjacent sites on DNA? If so, how do these lead to enhancement of transcription? The techniques to be used (and developed as needed) will be applicable to study of specific chromosomal proteins in mammalian organisms. The concepts which emerge from this work will influence future research on control processes in eukaryotic cells. For example, enhancer sequences in eukaryotes may function through direct contacts between proteins bound to different regions of DNA. Ultimately, the philosophy underlying this project is that a detailed understanding of normal regulatory processes is crucial to unravelling the mysteries of uncontrolled, malignant cell growth and of other pathological conditions as well.
