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High resolution contact probing of the Lrp‐like DNA‐binding protein Ss‐Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2
Author(s) -
EnoruEta Julius,
Gigot Daniel,
Glansdorff Nicolas,
Charlier Daniel
Publication year - 2002
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2002.03136.x
Subject(s) - sulfolobus solfataricus , biology , footprinting , binding site , dna , sulfolobus , dna footprinting , microbiology and biotechnology , base pair , biochemistry , homotetramer , heterotetramer , dna binding protein , biophysics , gene , transcription factor , protein subunit , archaea , base sequence
Summary 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.