title: Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase.
abstract: The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH-NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Tricia A
authors.initials: TA
authors.lastname: Windgassen
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Rachel Anne
authors.initials: RA
authors.lastname: Mooney
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Dhananjaya
authors.initials: D
authors.lastname: Nayak
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Murali
authors.initials: M
authors.lastname: Palangat
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Jinwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA landick@bact.wisc.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D014158:Transcription, Genetic
chemical_list: D009711:Nucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases