title: Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control.
abstract: Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Guo
authors.forename: C M
authors.initials: CM
authors.lastname: Lew
authors.forename: J D
authors.initials: JD
authors.lastname: Gralla
mesh_terms: D000225:Adenine / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D051922:PII Nitrogen Regulatory Proteins; D012313:RNA / Q000378:metabolism*; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D013696:Temperature; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D051922:PII Nitrogen Regulatory Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C497751:rpoN protein, E coli; D012313:RNA; D004247:DNA; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000225:Adenine