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DOI	10.1073/pnas.2115899118
	Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2
	Fukudome A.; Singh J.; Mishra V.; Reddem E.; Martinez-Marquez F.; Wenzel S.; Yan R.; Shiozaki M.; Yu Z.; Wang J.C.-Y.; Takagi Y.; Pikaard C.S.
发表日期	2021
ISSN	0027-8424
卷号	118 期号:51
英文摘要	RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV–RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 30 ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA’s 30 end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 30 end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 30 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Pol IV–RDR2 coupling; Polymerase backtracking; RDR2; RNA silencing; SiRNA biogenesis
语种	英语
scopus关键词	Arabidopsis protein; plant DNA; plant RNA; RDR2 protein, Arabidopsis; amino acid sequence; Arabidopsis; enzymology; gene expression regulation; genetic transcription; genetics; metabolism; molecular model; physiology; protein conformation; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; DNA, Plant; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Models, Molecular; Protein Conformation; RNA, Plant; RNA-Dependent RNA Polymerase; Transcription, Genetic
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/250920
作者单位	HHMI, Indiana University, Bloomington, IN 47405, United States; Department of Biology, Indiana University, Bloomington, IN 47405, United States; Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, United States; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 47405, United States; CryoEM Facility, Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, United States; Indiana University Electron Microscopy Center, Indiana University, Bloomington, IN 47405, United States
推荐引用方式 GB/T 7714	Fukudome A.,Singh J.,Mishra V.,et al. Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2[J],2021,118(51).
APA	Fukudome A..,Singh J..,Mishra V..,Reddem E..,Martinez-Marquez F..,...&Pikaard C.S..(2021).Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2.Proceedings of the National Academy of Sciences of the United States of America,118(51).
MLA	Fukudome A.,et al."Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2".Proceedings of the National Academy of Sciences of the United States of America 118.51(2021).