DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor.

Abstract

The affinity of the Eseherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center ofthe operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressoroperator complex. Central sequence effects on affinity for repressor are independent ofthe identity ofadjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.Our current picture of how the Escherichia coli phage 434 repressor recognizes its DNA operator is summarized in Fig. 1. In the repressor-operator complex, one a-helix of the repressor dimer lies in each half-site of the operator. Each of these helices is positioned in the major groove so that its side chains can contact the outermost 5 base pairs in one operator half-site (1). Biochemical and x-ray crystallographic studies suggest that neither this "recognition helix" nor any other part of the repressor contacts the innermost 2 base pairs of each half-site (1, 2). Nevertheless, the operator's affinity for repressor is determined, in part, by the composition of these base pairs: operators bearing APT or T'A base pairs at operator positions 6-9 bind the repressor more strongly than do operators bearing G-C or C-G base pairs at these positions (2). We have proposed that the base composition near the center of the operator affects its affinity for repressor by altering the ease with which operator DNA can be deformed into the optimal configuration for complex formation (2). Crystallographic analysis of the 434 repressor-operator complex shows that when 434 operator binds to 434 repressor, the DNA near the center of the operator is slightly overtwisted, so that the minor groove in this region is compressed (Fig. 1) (1). Evidently this DNA deformation is required to align the two operator half-sites so that each monomer of the bound repressor dimer can make optimal contacts with each half-site.In this paper we explore more directly the role of both DNA flexibility and repressor flexibility in determining the affinity of operator for repressor. We reasoned that introducing a single-strand break at the central phosphodiester bond of the operator would increase th...

In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation.

“…Mutations within a protein binding site but outside the direct protein-DNA contact regions may affect protein binding by influencing the DNA conformation, as shown for the phage 434 operator-repressor system (32,33). The conformational flexibility of the DNA is utilized to interact with proteins, and subsequently the DNA structure may get deformed (57).…”

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

“…Recognition of binding sites by DNA binding proteins such as 434 repressor, Cro, CAP, and other protein-DNA complexes has been proposed to be achieved by the sequence specific deformability of DNA (Drew & Travers, 1985;Crothers et al, 1988;Koudelka et al, 1988;Satchwell et al, 1986;Schultz et al, 1991). The bend in the DNA under consideration must be interpreted with respect to the location, magnitude, and direction of the bend introduced by the UvrB-DNA complex.…”

Enhanced and coordinated processing of synapsed viral DNA ends by retroviral integrases in vitro.

“…Many dimeric DNA-binding proteins that bind operators with two sites show cooperative binding and are also sensitive to the configuration between the DNA recognition sites. For example, the affinity of the phage 434 repressor for an operator can be increased by introducing a single-strand nick in the region linking two operator half-sites (Koudelka et al 1988). Measurements of the energetics of DNA twisting suggest that the single-stranded gaps between the binding sites can affect torsional relief to better accommodate protein binding (Koudelka and Carlson 1992).…”