DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Eσ70 as a cofactor for looping

Abstract

Transcription initiation by RNA polymerase (RNP) carrying the house-keeping σ subunit, σ⁷⁰ (Eσ⁷⁰), is repressed by H-NS at a number of promoters including hdeABp in Escherichia coli, while initiation with RNP carrying the stationary phase σ, σ³⁸ (Eσ³⁸), is not. We investigated the molecular mechanism of selective repression by H-NS to identify the differences in transcription initiation by the two forms of RNPs, which show indistinguishable promoter selectivities in vitro. Using hdeABp as a model promoter, we observed with purified components that H-NS, acting at a sequence centered at -118, selectively repressed transcription by Eσ⁷⁰. This selective repression is attributed to the differences in the interactions between hdeABp and the two forms of RNPs, since no other factor is required for the repression. We observed that the two forms of RNPs could form an open initiation complex (RP_O) at hdeABp, but that Eσ⁷⁰ failed to initiate transcription in the presence of H-NS. Interestingly, KMnO₄ assays and high-resolution atomic force microscopy (AFM) revealed that hdeABp DNA wrapped around Eσ⁷⁰ more tightly than around Eσ³⁸, resulting in the potential crossing over of the DNA arms that project out of Eσ⁷⁰ · RP_O but not out of Eσ³⁸ · RP_O. Based on these observations, we postulated that H-NS bound at -118 laterally extends by the cooperative recruitment of H-NS molecules to the promoter-downstream sequence joined by wrapping of the DNA around Eσ⁷⁰ · RP_O, resulting in effective sealing of the DNA loop and trapping of Eσ⁷⁰. Such a ternary complex of H-NS · Eσ⁷⁰hdeABp was demonstrated by AFM. In this case, therefore, Eσ⁷⁰ acts as a cofactor for DNA looping. Expression of this class of genes by Eσ³⁸ in the stationary phase is not due to its promoter specificity but to the architecture of the promoter · Eσ³⁸ complex.