title: Cys-pair reporters detect a constrained trigger loop in a paused RNA polymerase.
abstract: Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Dhananjaya
authors.initials: D
authors.lastname: Nayak
authors.forename: Michael
authors.initials: M
authors.lastname: Voss
authors.forename: Tricia
authors.initials: T
authors.lastname: Windgassen
authors.forename: Rachel Anne
authors.initials: RA
authors.lastname: Mooney
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D001483:Base Sequence; D003538:Cystamine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D055029:Inverted Repeat Sequences; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010445:Peptide Elongation Factors / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry*; D015534:Trans-Activators / Q000737:chemistry; D035602:Transcriptional Elongation Factors / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D010445:Peptide Elongation Factors; C106401:RfaH protein, E coli; D015534:Trans-Activators; D035602:Transcriptional Elongation Factors; D003553:Cystine; D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D003538:Cystamine