Metal A and metal B sites of nuclear RNA polymerases Pol IV and Pol V are required for siRNA-dependent DNA methylation and gene silencing.
Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active.
Haag
Jeremy R
JR
Department of Biology, Washington University, St Louis, MO, USA.
Pontes
Olga
O
Pikaard
Craig S
CS
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019175:DNA Methylation; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements; D016415:Sequence Alignment
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D008670:Metals; D021122:Protein Subunits; D034741:RNA, Small Interfering; D018626:Retroelements; C499181:RNA polymerase IV, Arabidopsis; D012321:DNA-Directed RNA Polymerases