Analysis of the DNA-binding sequence specificity of the archaeal transcriptional regulator Ss-LrpB from Sulfolobus solfataricus by systematic mutagenesis and high resolution contact probing.
To determine the sequence specificity of dimeric Ss-LrpB, a high resolution contact map was constructed and a saturation mutagenesis conducted on one half of the palindromic consensus box. Premodification binding interference indicates that Ss-LrpB establishes most of its tightest contacts with a single strand of two major groove segments and interacts with the minor groove at the center of the box. The requirement for bending is reflected in the preference for an A+T rich center and confirmed with C.G and C.I substitutions. The saturation mutagenesis indicates that major groove contacts with C.G at position 5 and its symmetrical counterpart are most critical for the specificity and strength of the interaction. Conservation at the remaining positions improved the binding. Hydrogen bonding to the O6 and N7 acceptor atoms of the G5' residue play a major role in complex formation. Unlike many other DNA-binding proteins Ss-LrpB does not establish hydrophobic interactions with the methyls of thymine residues. The binding energies determined from the saturation mutagenesis were used to construct a sequence logo, which pin-points the overwhelming importance of C.G at position 5. The knowledge of the DNA-binding specificity will constitute a precious tool for the search of new physiologically relevant binding sites for Ss-LrpB in the genome.
Peeters
Eveline
E
Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel and Vlaams interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussel, Belgium.
Wartel
Carine
C
Maes
Dominique
D
Charlier
Daniel
D
D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D006860:Hydrogen Bonding; D051756:Leucine-Responsive Regulatory Protein / Q000378:metabolism*; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D048229:Sulfolobus solfataricus
D019843:Archaeal Proteins; D051756:Leucine-Responsive Regulatory Protein; D004247:DNA