Repression by Binding of H-NS within the Transcription Unit

Abstract

H-NS inhibits transcription by forming repressing nucleoprotein complexes next to promoters. We investigated repression by binding of H-NS within the transcription unit using the bgl and proU operons. Repression of both operons requires a downstream regulatory element (DRE) in addition to an upstream element (URE). In bgl, H-NS binds to a region located between 600 to 700 bp downstream of the transcription start site, whereas in proU the DRE extends up to position ؉270. We show that binding of H-NS to the bgl-DRE inhibits transcription initiation at a step before open complex formation, as shown before for proU. This was shown by determining the occupancy of the bgl transcription unit by RNA polymerases, expression analysis of bgl and proU reporter constructs, and chloroacetaldehyde footprinting of RNA polymerase promoter complexes. The chloroacetaldehyde footprinting also revealed that RNA polymerase is "poised" at the osmoregulated 70-dependent proU promoter at low osmolarity, whereas at high osmolarity poising of RNA polymerase and repression by H-NS are reduced. Furthermore, repression by H-NS via the URE and DRE is synergistic, and the efficiency of repression by H-NS via the DRE inversely correlates with the promoter activity. Repression is high for a promoter of low activity, whereas it is low for a strong promoter. Inefficient repression of strong promoters by H-NS via a DRE may account for high induction levels of proU at high osmolarity and for bgl upon disruption of the URE.

Regulation of Expression of the Region 3 Promoter of theEscherichia coliK5 Capsule Gene Cluster Involves H-NS, SlyA, and a Large 5′ Untranslated Region

“…Both the proU and bgl operons have upstream regulatory elements (UREs) and DREs that are essential for H-NS repression (20). It has been demonstrated that binding of H-NS at the bgl DRE inhibits transcription initiation at a step prior to open complex formation (20) in a manner similar to that previously described for H-NS repression via the proU DRE (13). It is unclear whether H-NS repression via a DRE is a consequence of RNA polymerase (RNAP) being trapped in a closed complex or whether RNAP is excluded (20).…”

“…It has been demonstrated that binding of H-NS at the bgl DRE inhibits transcription initiation at a step prior to open complex formation (20) in a manner similar to that previously described for H-NS repression via the proU DRE (13). It is unclear whether H-NS repression via a DRE is a consequence of RNA polymerase (RNAP) being trapped in a closed complex or whether RNAP is excluded (20). A number of models have been proposed for the repression of transcription by H-NS via a DRE.…”

“…The importance of the binding of H-NS 3Ј of the transcription start site in temperature-dependent repression of PR3 is illustrated by plasmids pRSA-12 and pRSA-17, in both of which this 3Ј region is deleted and both of which have lost temperature regulation ( Table 3). The identification of H-NS binding sites 3Ј of the transcription start site has been seen with a number of H-NSregulated genes (7,17,21,22,28), of which the proU and bgl operons are best characterized (7,20). Both the proU and bgl operons have upstream regulatory elements (UREs) and DREs that are essential for H-NS repression (20).…”

“…The identification of H-NS binding sites 3Ј of the transcription start site has been seen with a number of H-NSregulated genes (7,17,21,22,28), of which the proU and bgl operons are best characterized (7,20). Both the proU and bgl operons have upstream regulatory elements (UREs) and DREs that are essential for H-NS repression (20). It has been demonstrated that binding of H-NS at the bgl DRE inhibits transcription initiation at a step prior to open complex formation (20) in a manner similar to that previously described for H-NS repression via the proU DRE (13).…”

Compensations for Diminished Terminal Oxidase Activity in Escherichia coli

“…In particular, hns transcription is down-regulated, and H-NS activity was shown to be diminished (Fig. 3); H-NS activity is diminished at high osmolarity (51). Two of the genes that are repressed by H-NS and induced by high osmolarity, bolA and osmC, are also upregulated in the microarray comparison (Figs.…”