title: RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.
abstract: Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
authors.forename: Monali
authors.initials: M
authors.lastname: NandyMazumdar
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nedialkov
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Svetlov
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Sevostyanova
authors.affiliation: Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
authors.forename: Georgiy A
authors.identifier: 0000-0002-3070-6843
authors.initials: GA
authors.lastname: Belogurov
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210; artsimovitch.1@osu.edu.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210.
authors.forename: Irina
authors.identifier: 0000-0002-8604-0980
authors.initials: I
authors.lastname: Artsimovitch
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D017353:Gene Deletion; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D010445:Peptide Elongation Factors / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012808:Sigma Factor / Q000737:chemistry*; D013824:Thermus / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D010445:Peptide Elongation Factors; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases
keywords: RNA polymerase; beta pincer; discriminator; promoter; transcription