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 transcription by σ⁵⁴-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between σ⁵⁴-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template). We show here that open complex formation is also thermodynamically unfavorable. In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones. Use of linear templates with a region of heteroduplex around the transcriptional start site-'preopened' templates-does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se. These results are in contrast 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 template or a preopened linear template.