Genetic (mutational) and biochemical methods will be used to investigate mechanisms of transcription initiation and its regulation in well-defined prokaryotic systems. Two promoters (transcription initiation signals) of bacteriophage lambdal will be studied in order to address the following questions: a) How do specific DNA sequences provide information for discrete steps in transcription initiation? b) What is the nature of the interaction between RNA polymerase and specific activator proteins that allows the activators to stimulate transcription initiation from weak promoters? c) How does the DNA sequence with which the polymerase interacts influence the interaction between the enzyme and a specific activator bound to an adjacent or overlapping recognition site? The specific aims of the proposed research are: (1) Use of oligonucleotide-directed mutagenesis to isolate specific mutations in the lambdal PRM promoter and evaluation of the effects of the mutations on promoter function in vitro. (2) Isolation of mutations in the lambdal cI protein--the specific activator of PRM--that would allow the protein to activate an altered promoter. (3) Isolation of mutations in the lambdal cII protein--the specific activator of PRE--that would affect the ability of the protein to activate PRE without affecting its ability to bind to DNA. (4) Kinetic analysis of the ability of lambdal cII protein to bind to wild-type PRE and PRE mutant DNA's. (5) Isolation of RNA polymerase mutants with altered ability to be activated by cII or cI protein. (6) Kinetic characterization of interactions of mutant proteins with their DNA recognition sites and with each other. These studies should contribute to an understanding of regulatory processes in higher organisms as well as in prokaryotes; they should help elucidate the nature of DNA sequence elements required for interaction with complex recognition proteins; and they should provide insights into protein-protein interactions that regulate transcription. The proposed research will be especially relevant to the ways that proteins interact at multiple regulatory sites in higher organisms to control gene activity in response to external stimuli.
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