Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS. Issue 21 (11th November 2014)

• Record Type: Journal Article
• Authors: Ulissi, Ulisse; Fabbretti, Attilio; Sette, Marco; Giuliodori, Anna Maria; Spurio, Roberto
• Is Part Of: Nucleic acids research. Volume 42:Issue 21(2014)
• Page Start: 13039
• Page End: 13050
• Publication Date: 2014-11-11
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gku1052
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract : The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NS ctd ), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The 'fast', 'intermediate' and 'slow' H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all 'fast' protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.