The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

H-NS is a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H-NS selectively silences specific genes of the bacterial chromosome. However, many studies have concluded that H-NS binds sequence-independently to DNA, leaving the molecular basis for its selectivity unexplained. We show that the negative regulatory element (NRE) of the supercoiling-sensitive Escherichia coliproU gene contains two identical high-affinity binding sites for H-NS. Cooperative binding of H-NS is abrogated by changes in DNA superhelical density and temperature. We further demonstrate that the high-affinity sites nucleate cooperative binding and establish a nucleoprotein structure required for silencing. Mutations in these sites result in loss of repression by H-NS. In this model, silencing at proU, and by inference at other genes directly regulated by H-NS, is tightly controlled by the cooperativity between bound H-NS molecules.

We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37°C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the ␣-subunit Cterminal domain and binding of the subunit to the ؊35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the ؊10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the ␤͞␤ jaws, leading to a transcriptionally active complex.DNA-protein interactions ͉ time-resolved footprinting ͉ transcription ͉ hydroxyl radicals D e novo RNA synthesis during DNA transcription is a highly regulated cellular process carried out by RNA polymerase. RNA polymerase binds to DNA and diffuses one-dimensionally to the promoter region (1-4); it then must make a number of interactions within the promoter as a prerequisite to forming a relatively stable complex. This complex is then in a state that favors isomerization, leading to strand separation from approximately Ϫ12 to ϩ3 with respect to the site of transcription initiation (5). Each of these steps is a possible site for regulation of the transcription levels. Extensive studies have been conducted on the structure of RNA polymerase-DNA complexes at equilibrium resulting in the characterization of the interaction of each of the enzyme's subunits with specific promoter elements (6, 7). However a direct characterization of the interactions formed at each step of the recognition process is still lacking.The T7A1 promoter used for the studies described here is one of the strongest known prokaryotic promoters (8). Its Ϫ35 sequence, TTGACT, is very close to the consensus (TTGACA), and its Ϫ10 sequence, GATACT, deviates from consensus (TATAAT). In addition, this promoter contains, from Ϫ42 to Ϫ80, a sequence rich in adenine and thymine residues, also known as an UP element (9-11). On other promoters this sequence has been shown to both increase the rate of promoter binding and stimulate isomerization to the open complex (12)(13)(14)(15). Kinetic studies on the formation of an RNA polymerasepromoter complex revealed the presence of a sequence of isomerization steps leading to the formation of an open complex (16-18). Furthermore, by decreasing the isomerization rates at lower temperatures, one or more intermediates in the pathway from the initial closed complex to the final open complex were isolated and characterized (19-22). However, the large, and sometimes nonlinear, temperature dependence of some ...