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-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.
Highlights
Enterobacterial histone-like nucleoid-structuring protein (H-NS) plays a dual role. It contributes to the architectural organization of the nucleoid and acts as a transcriptional repressor on a wide range of genes (∼5% of the total) [1,2,3,4,5], some encoding housekeeping functions, others implicated in cellular responses to environmental changes, including the virulence factors whose expression is triggered by the passage from the external environment to that of the host intestine [6,7]
Previous studies have demonstrated that H-NS targets two sites on the virF promoter: site I centered at −1 and site II centered at −250, separated by an intrinsic DNA curvature centered around −140 position [14]
The finding that the monomeric C-terminal domain of H-NS (HNSctd) displays the same binding selectivity as the native protein and yields well-defined footprints unlike the intact, oligomerizing H-NS [16,38] prompted a careful comparison of the preferred H-NS targets in different H-NS-sensitive promoters. This analysis led to the identification of short consensus sequences selectively recognized by H-NS; in turn, these sequences were suggested to represent high-affinity nucleation sites where the protein binds before building more complex nucleoprotein structures by spreading, through its oligomerization, along lower affinity secondary sites [22,39]
Summary
Enterobacterial histone-like nucleoid-structuring protein (H-NS) plays a dual role. It contributes to the architectural organization of the nucleoid and acts as a transcriptional repressor on a wide range of genes (∼5% of the total) [1,2,3,4,5], some encoding housekeeping functions, others implicated in cellular responses to environmental changes, including the virulence factors whose expression is triggered by the passage from the external environment to that of the host intestine [6,7].Different mechanisms, such as (i) promoter occlusion, as postulated for hns autorepression [8,9,10] and (ii) RNA polymerase entrapment in the promoter caused by H-NSmediated DNA looping [11,12] have been implicated in transcriptional repression by H-NS.The Shigella plasmid-encoded virF encodes the activator of the multistep pathogenicity cascade of this enterobacterium and is among the virulence genes subjected to temperature-dependent transcriptional repression by H-NS [13,14]. Enterobacterial histone-like nucleoid-structuring protein (H-NS) plays a dual role It contributes to the architectural organization of the nucleoid and acts as a transcriptional repressor on a wide range of genes (∼5% of the total) [1,2,3,4,5], some encoding housekeeping functions, others implicated in cellular responses to environmental changes, including the virulence factors whose expression is triggered by the passage from the external environment to that of the host intestine [6,7]. Previous studies have demonstrated that H-NS targets two sites on the virF promoter: site I centered at −1 and site II centered at −250, separated by an intrinsic DNA curvature centered around −140 position [14].
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