title: In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing.
abstract: In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases Pol IV and Pol V and the putative RNA-dependent RNA polymerase RDR2. Here we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to α-amanitin, and differ in their ability to displace the nontemplate DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is nonfunctional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.
authors.affiliation: Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Jeremy R
authors.initials: JR
authors.lastname: Haag
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Ream
authors.forename: Michelle
authors.initials: M
authors.lastname: Marasco
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.forename: Angela D
authors.initials: AD
authors.lastname: Norbeck
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolic
authors.forename: Craig S
authors.initials: CS
authors.lastname: Pikaard
mesh_terms: D053959:Alpha-Amanitin / Q000494:pharmacology; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000096:biosynthesis*; D018749:RNA, Plant / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D053959:Alpha-Amanitin; D029681:Arabidopsis Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012330:RNA, Double-Stranded; D018749:RNA, Plant; D034741:RNA, Small Interfering; D004247:DNA; C499181:RNA polymerase IV, Arabidopsis; C538841:RNA polymerase V, Arabidopsis; C519655:RDR2 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases