The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin.
Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.
Department of Structural Biology, Stanford University, Stanford, CA 94305, USA. cdkaplan@stanford.edu
Craig D
CD
Kaplan
Karl-Magnus
KM
Larsson
Roger D
RD
Kornberg
D053959:Alpha-Amanitin / Q000494:pharmacology*; D019943:Amino Acid Substitution; D004251:DNA Transposable Elements / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010641:Phenotype; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects*
D053959:Alpha-Amanitin; D004251:DNA Transposable Elements; D019384:Nucleic Acid Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae