THE BACTERIAL ENHANCER-BINDING PROTEIN NTRC IS A MOLECULAR MACHINE - ATP HYDROLYSIS IS COUPLED TO TRANSCRIPTIONAL ACTIVATION

NTRC is a prokaryotic enhancer-binding protein that activates transcri ption by sigma(54)-holoenzyme. NTRC has an ATPase activity that is req uired for transcriptional activation, specifically for isomerization o f closed complexes between sigma(54)-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a k inetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts e ven from a supercoiled template). We show here that open complex forma tion is also thermodynamically unfavorable, In the absence of ATP hydr olysis the position of equilibrium between closed and open complexes f avors the closed ones. Use of linear templates with a region of hetero duplex around the transcriptional start site-''preopened'' templates-d oes not bypass the requirement for either NTRC or ATP hydrolysis, prov iding evidence that the rate-limiting step in open complex formation d oes not lie in DNA strand denaturation per se. These results are in co ntrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid t emplate or a preopened linear template.