The objective of this research is to identify protein-DNA and protein-protein interactions that regulate the assembly of the prokaryotic transcriptional initiation complex, and to gain and understanding of their roles in the control of its activity. The functions of the E. coli RNA polymerase, lactose (lac) repressor and cyclic AMP receptor protein (CAP) at the lactose promoter-operator region will be investigated. The individual activities of these proteins within this region have been the subjects of a significant amount of research, and are well characterized. What is lacking at present is an understanding of how they interact to regulate transcription. To address this crucial issue, studies will be carried out to: 1. determine the effects of each protein on the lac promoter binding affinities, stoichiometries and sites of interaction of each of the others; 2. measure the effects of each protein on the association and dissociation kinetics of each of others; 3. characterize the transcriptional activity of each complex containing RNA polymerase in terms of: (a) isomerization state- whether "open" or "closed"; (b) its abortive initiation kinetics; (c) the ratio of abortive to productive initiation and (d) the ratio of transcriptional initiation at each of the two overlapping lac promoters (P1 and P2). 4. determine the effects of extra-promoter regulatory sequences on the composition, stability and activity of complexes of CAP, lac repressor and RNA polymerase at the lac promoter in vitro and on the induction of transcription in vivo. Because each protein has at least two high affinity binding sites within the lac promoter, several different binding arrangements and transcriptional activity states may exist for a given binding stoichiometry. For this reason, specific aims 1 and 3 will be carried out in a concerted manner, allowing correlation of the protein stoichiometry, binding site occupancy and transcriptional activity of each complex. //
