RAMAN SPECTRAL STUDIES OF NUCLEIC-ACIDS .42. DNA RECOGNITION BY THE HELIX-TURN-HELIX MOTIF - INVESTIGATION BY LASER RAMAN-SPECTROSCOPY OF THE PHAGE LAMBDA REPRESSOR AND ITS INTERACTION WITH OPERATOR SITES OL1 AND OR3

The lambda-repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda-operator sites O(L)1 and O(R)3 and their interaction with the DNA-binding domain of X repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous O(L)1 and O(R)3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda-domain is not greatly perturbed by binding to either O(L)1 or O(R)3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the O(L)1 complex; yet, Y22 hydrogen bonding is not altered in forming the O(R)3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the X repressor DNA-binding domain with operators O(L)1 and O(R)3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.