Deuterium exchange of operator 8CH groups as a Raman probe of repressor recognition: interactions of wild-type and mutant .lambda. repressors with operator OL1

• Published in: Biochemistry (Easton) Vol. 31; no. 12; pp. 3118 - 3125
• Main Authors: Reilly, Kim E, Becka, Renee, Thomas, George J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 31.03.1992
• Subjects: Adenine - chemistry; Adenine Nucleotides - chemistry; Amino Acid Sequence; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Deuterium - chemistry; DNA Probes - chemistry; DNA-Binding Proteins; Fundamental and applied biological sciences. Psychology; Guanine - chemistry; Guanine Nucleotides - chemistry; Kinetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Operator Regions, Genetic; Purines - chemistry; Repressor Proteins - chemistry; Repressor Proteins - genetics; Spectrum Analysis, Raman; Temperature; Transcription Factors - genetics; Transcription. Transcription factor. Splicing. Rna processing; Viral Proteins; Viral Regulatory and Accessory Proteins; Siphoviridae; Transcription repressor; Molecular exchange; Virus; Site specificity; Transcription operator; DNA; Molecular complex; Purine; Raman spectrometry; Phage lambda; Recognition; Phage
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00127a012

The rate of deuterium exchange of a purine 8CH group in DNA is highly sensitive to both macromolecular secondary structure and intermolecular interactions which restrict solvent access to the major groove [Lamba, O.P., Becka, R., & Thomas, G.J., Jr. (1990) Biopolymers 29, 1465-1477]. We have exploited the sensitivity of the 8CH---8CD reaction to probe DNA recognition by the helix-turn-helix (HTH) motif of phage lambda cI repressor. We find that purine exchanges in the 19-base-pair OL1 operator are strongly and specifically restricted by binding of the HTH N-terminal domain of the repressor fragment (RF) comprising residues 1-102. The kinetics indicate large-scale obstruction of solvent access to operator 7N-8C purine sites. Interpretation of the exchange kinetics using a simple model suggests that only 7 purine residues (5 of 10 adenines and 2 of 9 guanines) remain unrestricted with respect to 8CH exchange in complexes of OL1 with the wild-type repressor. On the other hand, the 8CH exchange profile for the complex of OL1 with the Tyr88---Cys mutant repressor indicates that 9 purines (7 adenines and 2 guanines) are exchangeable. These results suggest important differences in major groove recognition in the two complexes. The proposed 8CH labeling profiles are consistent with molecular models of related complexes determined by X-ray crystallography [Jordan, S.R., & Pabo, C.O. (1988) Science 242, 893-899] and indicate that the structures observed in the crystal are largely maintained in solution.