High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2.
Ss-Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine-responsive regulatory protein), which out of all genome-encoded proteins is most similar to Escherichia coli Lrp (E-value of 5.6 e-14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss-Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss-Lrp binding was established by DNase I and hydroxyl radical footprinting, small non-intercalating groove-specific ligand-binding interference, and various base-specific premodification and base removal binding interference techniques. We show that Ss-Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA-box and BRE promoter elements. Therefore, Ss-Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss-Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019848:Gene Expression Regulation, Archaeal; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D016944:Sulfolobus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein