Genetic (mutational) and biochemical methods will be combined in the investigation of mechanisms of transcription initiation and promoter site selection in well-defined prokaryotic systems. Several promoters (transcription initiation sites) will be studied in order to address the following questions: a) What is the relationship between nucleotide sequences in promoter regions and specific steps in transcription initiation? b) How do specific DNA sequences determine whether a promoter will be a strong or weak signal for transcription initiation? c) How can positive regulatory proteins change a weak signal into a strong signal? The specific aims of the proposed research are: (1) Isolation of new mutations in the P(RM) and P(R) promoters of bacteriophage lambda by site-specific mutagenesis in vitro. (2) Kinetic analysis of the mutant promoters to determine how specific mutations affect kinetically distinguishable steps in transcription initiation. (3) Kinetic analysis of existing mutations in the LambdaP(RE) promoter to determine which steps in initiation are affected by mutation, and to characterize mutations that cause defects in activation of P(RE) by Lambda cII protein. (4) Isolation of mutations in the Lambda P'(R) promoter. (5) Isolation of mutations in the Gal P2 promoter to facilitate investigation of the role of P2 in expression of the bacterial galactose operon. These studies should contribute to an understanding of regulatory processes in both prokaryotes and higher organisms, and should help elucidate the nature of DNA sequence elements required for interaction with complex recognition proteins. In addition, the proposed research should provide new information about how the frequency of gene expression can be altered by mutations in promoter DNA sequences and by the action of accessory regulatory proteins.
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