Abstract
H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has been shown to exhibit a binding preference for certain AT-rich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A Kd of 1-2 microM was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.
Highlights
H-NS is a 15.6-kDa polypeptide, one of the two most abundant proteins associated with the bacterial nucleoid
To address the mechanisms by which H-NS affects transcription we have used the proU promoter of Salmonella typhimurium as a model. proU encodes a transport system for the osmoprotectant glycine betaine, and its expression is increased when cells are exposed to high extracellular osmolarity (20 – 23). proU appears to be one of the simplest H-NS-responsive promoters as no trans-acting factor, other than H-NS itself, appears to be required for its regulation. proU expression is derepressed in hns mutants [6, 7, 24], residual osmoregulation is retained [13, 25, 26]
Previous studies have shown that H-NS exhibits a binding preference for curved DNA resulting from phased A tracts
Summary
Bacterial Strains and Plasmids—Wild-type S. typhimurium LT2 and its congenic derivatives CH1838 (hns-1::Kanr) and CH1833 (zde-1710::mTn10) were used for expression studies [8]. A sixth plasmid (pAF6) was constructed by ligating a 200-bp fragment encompassing the S. typhimurium proU DRE (base pair ϩ99 through ϩ299; Ref. 30) between the NotI and SpeI sites of pBluescript II SK(Ϫ). To clone these curved/noncurved sequences between the proU promoter and the lacZ reporter gene, a multiple cloning site was created in plasmid pAV399 [13]. Plasmids with Altered Distances between the proU Promoter and the Downstream Curve—pSJ4 contains the same 317-bp proU promoter fragment (bp Ϫ217 to ϩ100) as pAV399, cloned into the EcoRI site upstream of the luciferase reporter genes in pSB71 (Table II). Appropriate curved or noncurved DNA fragments were isolated from plasmids by restriction enzyme digestion and gel purification.
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