Open AccessTime-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS
Author(s) -
Ulisse Ulissi,
Attilio Fabbretti,
Marco Sette,
Anna Maria Giuliodori,
Roberto Spurio
Publication year - 2014
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gku1052
Subject(s) - biology , dna footprinting , repressor , nucleoprotein , footprinting , binding site , derepression , microbiology and biotechnology , promoter , dna , shigella flexneri , nucleoid , psychological repression , transcription (linguistics) , deoxyribonuclease i , dna binding protein , dna binding site , transcription factor , gene , genetics , escherichia coli , gene expression , philosophy , base sequence , linguistics
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-NSctd), 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.