The mechanism of DNA-operator recognition by repressor and cro proteins of bacteriophage lambda, 434 and P22 will be analyzed by a combination of crystallographic structural studies and directed mutagenesis. The structure of a cocrystal of the amino-terminal domain of 434 repressor with its complete 14 base pair operator will be determined to about 3 A (the limit of diffraction) using standard crystallographic methods. Directed mutagenesis will be used to generate modified 434 and lambda repressors and cros in order to test and extend models for recognition based on the crystal structure. The binding specificities and regulatory properties of the altered proteins will be examined. Efforts to crystallize some of the modified proteins, as well as to crystallize 434 repressor alone and 434 cro with and without DNA will be made, since these structures are essential to a complete picture of operator binding. Modified operators will likewise be constructed. Mechanisms of positive control in lambda will be studied by directing changes in residues implicated in repressor-polymerase contact by pc mutations, and structures of potentially informative modified repressors will be studied by difference Fourier analysis when possible. This group of proteins presents an outstanding opportunity for understanding regulatory interactions of the proteins with each other and with DNA. We anticipate that, ultimately, it will be possible to create repressors with entirely new specificities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029109-06
Application #
3276598
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1981-04-01
Project End
1989-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
6
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Shimon, L J; Harrison, S C (1993) The phage 434 OR2/R1-69 complex at 2.5 A resolution. J Mol Biol 232:826-38
Rodgers, D W; Harrison, S C (1993) The complex between phage 434 repressor DNA-binding domain and operator site OR3: structural differences between consensus and non-consensus half-sites. Structure 1:227-40
Mondragon, A; Harrison, S C (1991) The phage 434 Cro/OR1 complex at 2.5 A resolution. J Mol Biol 219:321-34
Pabo, C O; Aggarwal, A K; Jordan, S R et al. (1990) Conserved residues make similar contacts in two repressor-operator complexes. Science 247:1210-3
Mondragon, A; Subbiah, S; Almo, S C et al. (1989) Structure of the amino-terminal domain of phage 434 repressor at 2.0 A resolution. J Mol Biol 205:189-200
Mondragon, A; Wolberger, C; Harrison, S C (1989) Structure of phage 434 Cro protein at 2.35 A resolution. J Mol Biol 205:179-88
Koudelka, G B; Harbury, P; Harrison, S C et al. (1988) DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor. Proc Natl Acad Sci U S A 85:4633-7
Hollis, M; Valenzuela, D; Pioli, D et al. (1988) A repressor heterodimer binds to a chimeric operator. Proc Natl Acad Sci U S A 85:5834-8
Harrison, S C; Anderson, J E; Koudelka, G B et al. (1988) Recognition of DNA sequences by the repressor of bacteriophage 434. Biophys Chem 29:31-7
Aggarwal, A K; Rodgers, D W; Drottar, M et al. (1988) Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science 242:899-907

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