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 OL1 and OR3 and their interaction with the DNA-binding domain of lambda 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 OL1 and OR3 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 OL1 or OR3, 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 OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 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.
Benevides
J M
JM
Division of Cell Biology and Biophysics, School of Basic Life Sciences, University of Missouri--Kansas City 64110.
Weiss
M A
MA
Thomas
G J
GJ
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D007834:Lasers; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013059:Spectrum Analysis, Raman / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA