The goal of this research program is to elucidate physical mechanisms of gene control processes mediated by the interactions of proteins at specific sites on DNA. Components of the program include: (a) development of new techniques for the study of site-specific protein-DNA interactions, (b) experimental studies of functional energetics in specific systems, (c) correlation of the energetic properties with structural features of the interacting molecules, and (d) development and testing of physical- chemical models for gene control. We plan to extend our previous studies on the repressor-operator control system of bacteriophage lambda, which is a prototype for a large family of cooperative multi-site gene control systems. Using techniques of calorimetry in combination with quantitative DNase footprint titration and a new cryogenic gel shift technique that we have developed, we will carry out a detailed study of the effects of temperature, pH, and salt interactions on the site-specific energetics (including cooperativity effects) of cI and cro repressor binding to the DNA operator sites. The results will provide new insights into the detailed roles of proton binding, salt binding, and the various non-covalent forces responsible for the regulatory interactions. These studies will be extended to a series of mutant repressors that are deficient in cooperativity. We will study the biological role of cooperative interactions between operator-bound repressors by in vitro and in vivo studies of mutant repressors that have altered cooperativity, using a physical-chemical model of gene control that is being developed under this program of research.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039343-09
Application #
2518939
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-07-01
Project End
1999-08-31
Budget Start
1997-09-01
Budget End
1999-08-31
Support Year
9
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Darling, P J; Holt, J M; Ackers, G K (2000) Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations. Biochemistry 39:11500-7
Darling, P J; Holt, J M; Ackers, G K (2000) Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers. J Mol Biol 302:625-38
Merabet, E K; Burz, D S; Ackers, G K (1998) Thermal melting properties of C-terminal domain mutants of bacteriophage lambda cI repressor. Methods Enzymol 295:450-67
Pray, T R; Burz, D S; Ackers, G K (1998) Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis. J Mol Biol 282:947-58
Bain, D L; Ackers, G K (1998) A quantitative cryogenic gel-shift technique for analysis of protein-DNA binding. Anal Biochem 258:240-5
Burz, D S; Ackers, G K (1996) Cooperativity mutants of bacteriophage lambda cI repressor: temperature dependence of self-assembly. Biochemistry 35:3341-50
Merabet, E; Ackers, G K (1995) Calorimetric analysis of lambda cI repressor binding to DNA operator sites. Biochemistry 34:8554-63
Bain, D L; Ackers, G K (1994) Self-association and DNA binding of lambda cI repressor N-terminal domains reveal linkage between sequence-specific binding and the C-terminal cooperativity domain. Biochemistry 33:14679-89
Burz, D S; Beckett, D; Benson, N et al. (1994) Self-assembly of bacteriophage lambda cI repressor: effects of single-site mutations on the monomer-dimer equilibrium. Biochemistry 33:8399-405
Burz, D S; Ackers, G K (1994) Single-site mutations in the C-terminal domain of bacteriophage lambda cI repressor alter cooperative interactions between dimers adjacently bound to OR. Biochemistry 33:8406-16

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