DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Transcription from many bacterial promoters is sensitive to the level of DNA supercoiling. We have investigated the mechanism by which environmentally induced changes in DNA supercoiling might regulate transcription. For the proU promoter of Salmonella typhimurium, osmotically induced changes in DNA topology appear to play a primary regulatory role. Changes in DNA supercoiling (linking number; delta Lk) are partitioned into changes in the winding of the strands of the double helix about themselves (twist; delta Tw) and/or elastic deformations or flexibility of the DNA helix (writhe; delta Wr). Mutations of the proU promoter were isolated in vivo, or generated in vitro, which altered the spacing between the -10 and -35 motifs. Studies on these mutant promoters, both in vivo and in vitro, exclude models in which changes in DNA twist play a regulatory role. Instead, our data suggest that increased DNA flexibility, reflecting the osmotically induced increase in negative supercoiling of DNA, is required for promoter activation.

Stimulation of transcription factor binding and histone displacement by nucleosome assembly protein 1 and nucleoplasmin requires disruption of the histone octamer.

To investigate the mechanisms by which transcription factors invade nucleosomal DNA and replace histones at control elements, we have examined the response of the histone octamer to transcription factor binding in the presence of histone-binding proteins (i.e., nucleosome assembly factors). We found that yeast nucleosome assembly protein 1 (NAP-1) stimulated transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. In addition, disruption of the histone octamer was required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP-1. While NAP-1 and nucleoplasmin stimulated the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation was lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP-1 and nucleoplasmin were able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity was also forfeited when the histone octamers were cross-linked. These data indicate that octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP-1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA did not require disruption of the histone octamer. Thus, histone displacement in this instance occurred by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP-1.

The chromatin-associated protein H-NS alters DNA topology in vitro.

H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.

The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression.

H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression.