Deuterium exchange of operator 8CH groups as a Raman probe of repressor recognition: interactions of wild-type and mutant lambda repressors with operator OL1.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Reilly
K E
KE
Division of Cell Biology and Biophysics, School of Basic Life Sciences, University of Missouri-Kansas City 64110.
Becka
R
R
Thomas
G J
GJ
D000225:Adenine / Q000737:chemistry*; D000227:Adenine Nucleotides / Q000737:chemistry; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry*; D004268:DNA-Binding Proteins; D003903:Deuterium / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011687:Purines / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D000227:Adenine Nucleotides; D015342:DNA Probes; D004268:DNA-Binding Proteins; D006150:Guanine Nucleotides; D011687:Purines; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D006147:Guanine; D003903:Deuterium; D000225:Adenine