High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2.

Abstract

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.