Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R. capsulatus sigma 70 RNA polymerase, but does not activate the Escherichia coli sigma 70-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 Rc alpha beta beta' or hybrid RNA polymerases containing the Rc beta beta' subunits absolutely require the inclusion of an omega subunit, with the Ec omega subunit only partially promoting RNA polymerase assembly. The Rc alpha Ec beta beta' RNA polymerase is not activated by RcNtrC. Moreover, a mutant form of the Rc alpha lacking the alpha C-terminal domain, when assembled with the Rc beta beta'omega and sigma 70 subunits, is activated by RcNtrC. These results suggest that the R. capsulatus alpha subunit is not important for RcNtrC interaction. All hybrid RNA polymerases that contained the Rc beta' were activated by RcNtrC, suggesting that the Rc beta' subunit plays an important role. It is proposed that RcNtrC recruits R. capsulatus sigma 70-RNA polymerase to the promoter through interaction with Rc beta'. 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.