Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12

Abstract

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of 'constitutive' promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ⁷⁰), RpoS (σ³⁸), RpoH (σ³²), RpoF (σ²⁸) or RpoE (σ²⁴), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ⁵⁴), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61-81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the 'repressive' promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

Keywords: Escherichia coli; NtrC; RNA polymerase; RpoN sigma factor; gSELEX; nitrogen metabolism.