¹H NMR sudy of the interaction of bacteriophageλ Cro protein with the O_R3 operator. Evidence for a change of the conformation of the O_R3 operator on binding

Abstract

The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.

DNA bending induced by Cro protein binding as demonstrated by gel electrophoresis.

“…Earlier NMR results (8) have suggested that the bend is near the middle of the OR3 region, at the GC dinucleotide pair at position 9. More recent data (10) have placed the bend into two sites several bases away from the pseudodyad center.…”

“…NMR studies can, in principle, also obtain detailed information not only on specific protein-DNA contacts but also on binding-induced DNA and protein structural distortion, and a number of recent studies have directly addressed these questions (5,6,(8)(9)(10). Comparative gel electrophoresis techniques have also been used to obtain information on proteininduced DNA bending in solution.…”

Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex.

“…This is about 2.9 A less than would be anticipated for straight B-DNA. NMR studies of Cro-OR3 complexes (22)(23)(24) have indicated bending or distortion near the center of the DNA and theoretical calculations (25) suggest that electrostatic interactions favor Cro binding to a bent form of DNA.…”

Structures and related properties of helical, disulfide-stabilized peptides

“…· . and A Repressor are bent at the junction of the two half-sites, but each half-site conforms to B-form DNA when bound to these repressors (Kirpichnikov, et al, 1984;Metzler & Lti, 1989;Evertsz, et al, 1991;Brennan, et al, 1986;Jordan & Pabo, 1988). A comparison Figure 4.12.…”