title: Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues.
abstract: The trigger loop (TL) of RNA polymerase II (Pol II) is a conserved structural motif that is crucial for Pol II catalytic activity and transcriptional fidelity. The TL remains in an inactive open conformation when the mismatched substrate is bound. In contrast, TL switches from an inactive open state to a closed active state to facilitate nucleotide addition upon the binding of the cognate substrate to the Pol II active site. However, a comprehensive understanding of the specific chemical interactions and substrate structural signatures that are essential to this TL conformational change remains elusive. Here we employed synthetic nucleotide analogues as 'chemical mutation' tools coupling with α-amanitin transcription inhibition assay to systematically dissect the key chemical interactions and structural signatures governing the substrate-coupled TL closure in Saccharomyces cerevisiae Pol II. This study reveals novel insights into understanding the molecular basis of TL conformational transition upon substrate binding during Pol II transcription. This synthetic chemical biology approach may be extended to understand the mechanisms of other RNA polymerases as well as other nucleic acid enzymes in future studies.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
authors.forename: Kyle Vincent
authors.initials: KV
authors.lastname: Butler
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Chong
authors.affiliation: Nucleic Acid Center and Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA dongwang@ucsd.edu.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
mesh_terms: D053959:Alpha-Amanitin / Q000737:chemistry; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D007700:Kinetics; D009696:Nucleic Acids / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014158:Transcription, Genetic
chemical_list: D053959:Alpha-Amanitin; D009696:Nucleic Acids; D009841:Oligonucleotides; D012330:RNA, Double-Stranded; D029701:Saccharomyces cerevisiae Proteins; C477371:locked nucleic acid; D012319:RNA Polymerase II