Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.
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.
Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC) 62032, Italy.
Ulisse
U
Ulissi
Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC) 62032, Italy.
Attilio
A
Fabbretti
Department of Chemical Sciences and Technologies, University of Rome-Tor Vergata, 00133 Roma, Italy.
Marco
M
Sette
Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC) 62032, Italy annamaria.giuliodori@unicam.it.
Anna Maria
AM
Giuliodori
Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC) 62032, Italy roberto.spurio@unicam.it.
Roberto
R
Spurio
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D012763:Shigella flexneri / Q000235:genetics* / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012097:Repressor Proteins; D037521:Virulence Factors