title: Wide-ranging and unexpected consequences of altered Pol II catalytic activity in vivo.
abstract: Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin immunoprecipitation can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants' growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Indranil
authors.initials: I
authors.lastname: Malik
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Chenxi
authors.initials: C
authors.lastname: Qiu
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Snavely
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Craig D
authors.initials: CD
authors.lastname: Kaplan
mesh_terms: D055162:Biocatalysis; D047369:Chromatin Immunoprecipitation; D015966:Gene Expression Regulation, Fungal; D006207:Half-Life; D009154:Mutation; D009711:Nucleotides / Q000172:deficiency; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D061805:Transcription Elongation, Genetic; D024363:Transcription Initiation Site
chemical_list: D009711:Nucleotides; D012333:RNA, Messenger; D012319:RNA Polymerase II