title: The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation.
abstract: NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-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.
authors.affiliation: Department of Plant Biology, University of California Berkeley 94720, USA.
authors.forename: A
authors.initials: A
authors.lastname: Wedel
authors.forename: S
authors.initials: S
authors.lastname: Kustu
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D009876:Operon; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012486:Salmonella typhimurium / Q000302:isolation & purification / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase