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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI017508-09
Application #
3127253
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-12-01
Project End
1992-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Arts and Sciences
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Fong, R S; Woody, S; Gussin, G N (1994) Direct and indirect effects of mutations in lambda PRM on open complex formation at the divergent PR promoter. J Mol Biol 240:119-26
Auerbach, S; Gao, J; Gussin, G N (1993) Nucleotide sequences of the trpI, trpB, and trpA genes of Pseudomonas syringae: positive control unique to fluorescent pseudomonads. Gene 123:25-32
Fong, R S; Woody, S; Gussin, G N (1993) Modulation of P(RM) activity by the lambda PR promoter in both the presence and absence of repressor. J Mol Biol 232:792-804
Woody, S T; Fong, R S; Gussin, G N (1993) Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter. Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR. J Mol Biol 229:37-51
Woody, S T; Fong, R S; Gussin, G N (1993) A cryptic promoter in the O(R) region of bacteriophage lambda. J Bacteriol 175:5648-54
Gussin, G N; Olson, C; Igarashi, K et al. (1992) Activation defects caused by mutations in Escherichia coli rpoA are promoter specific. J Bacteriol 174:5156-60
Gao, J; Gussin, G N (1991) Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. EMBO J 10:4137-44
Gao, J G; Gussin, G N (1991) RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins. J Bacteriol 173:394-7
Gao, J G; Gussin, G N (1991) Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro. J Bacteriol 173:3763-9
Brown, S; Ferm, J; Woody, S et al. (1990) Selection for mutations in the PR promoter of bacteriophage lambda. Nucleic Acids Res 18:5961-7

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