Using genetic biochemical and physical methods, we will examine the mechanism by which the noncontacted bases in the lac, phage P22 and phage 434 operators affect affinity for lac repressor, P22 repressor and Cro, and 434 repressor and Cro proteins, respectively. (i) 434 repressor and Cro. To substantiate our claim that it is the resistance of the central base pairs in the operator that determines the affinity of the operator for these proteins we will show that: (a) the twist of the free DNA's and uncorrelated with affinity; (b) 434 repressor and Cro overwind DNA and that all operators are overwound to the same extent; (c) a chemically modified operator that is overwound prior to interacting with the proteins, binds with a much higher affinity to the proteins than an unmodified operator of identical sequence. The role of the repressor and Cro's dimer interfaces in determining the central operator sequence specificity will be investigated by both random and site-directed mutations. (ii) P22 repressor and Cro. Circumstantial evidence suggests that, like 434 repressor, P22 repressor and Cro overwind the DNA at the center of the operator. We will determine the central sequence preferences of P22 repressor and Cro and compare them to those of 434 repressor and Cro. Subsequently, we will test whether the resistance of the central base pairs to overtwisting plays a role in the sequence specificity of P22 repressor and Cro. (iii) Lac repressor. Lac repressor appears to unwind DNA as it binds. We will characterize the DNA sequences that facilitate or inhibit the formation of the lac repressor-operator complex. By combining the information on central sequence preferences of these proteins, we will gain insight into the structure and sequence determinants of DNA structure and flexibility. This information will be useful in interpreting the role of DNA sequence in terms of effects on structure and flexibility in many other systems of biological interest.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29GM042138-01
Application #
3467640
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-04-01
Project End
1994-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260