RNA polymerase subunit requirements for activation by the enhancer-binding protein Rhodobacter capsulatus NtrC

Abstract

Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R. capsulatus σ70 RNA polymerase, but does not activate the Escherichia coli σ70-RNA polymerase at the nifA1 promoter. We utilized R. capsulatus:E. coli hybrid RNA polymerases assembled in vitro to investigate the subunits required for protein-protein interaction with RcNtrC at the nifA1mut1 promoter. Assembly of core Rcαββ′ or hybrid RNA polymerases containing the Rcαββ′ subunits absolutely require the inclusion of an ω subunit, with the Ecω subunit only partially promoting RNA polymerase assembly. The RcαEcβ′ RNA polymerase is not activated by RcNtrC. Moreover, a mutant form of the Rcα lacking the α C-terminal domain, when assembled with the Rcββ′ ω and σ70 subunits, is activated by RcNtrC. These results suggest that the R. capsulatus α subunit is not important for RcNtrC interaction. All hybrid RNA polymerases that contained the Rcβ′ were activated by RcNtrC, suggesting that the Rcβ′ subunit plays an important role. It is proposed that RcNtrC recruits R. capsulatus σ70-RNA polymerase to the promoter through interaction with Rcβ′. RcNtrC interacts with RNA polymerase from a unique position, with dimers centered at -118 bp from the start site. Placing the RcNtrC tandem binding sites on the opposite face of the helix (-113 bp) completely abolished transcription activation. Moving the RcNtrC tandem binding sites 20 bp closer to or further from the promoter significantly reduced activation, again suggesting unique spatial constraints on how RcNtrC interacts with the R. capsulatus RNA polymerase.