|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|Peeters, E.; Wartel, C.; Maes, D.; Charlier, D. (2007). 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. Nucleic Acids Res. 35(2): 623-633. dx.doi.org/10.1093/nar/gkl1095
In: Nucleic acids research. Information Retrieval: London. ISSN 0305-1048, more
|Authors|| || Top |
- Peeters, E.
- Wartel, C.
- Maes, D.
- Charlier, D.
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 O-6 and N-7 acceptor atoms of the G(5') 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.