与水稻中内源性双链RNA相关的RNA依赖性RNA聚合酶活性

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice.

作者信息

Horiuchi H, Udagawa T, Koga R, Moriyama H, Fukuhara T

机构信息

Laboratory of Molecular and Cellular Biology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo, 183-8509 Japan.

出版信息

Plant Cell Physiol. 2001 Feb;42(2):197-203. doi: 10.1093/pcp/pce025.

DOI:10.1093/pcp/pce025

PMID:11230574

Abstract

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.

摘要

在含有14千碱基对双链RNA（dsRNA）的水稻培养细胞的粗微粒体部分中检测到了RNA依赖性RNA聚合酶（RdRp）活性。RdRp活性在所有四种核苷三磷酸和Mg2+离子存在的情况下最大，并且对DNA依赖性RNA聚合酶的抑制剂（放线菌素D和α-鹅膏蕈碱）具有抗性。在0.5%脱氧胆酸盐存在的情况下，RdRp活性增加约2.5倍。用蛋白酶K加脱氧胆酸盐处理纯化的微粒体部分表明，与其自身14 kb RNA模板结合的RdRp酶复合物位于囊泡中。RdRp酶复合物用Nonidet P-40增溶并通过甘油梯度离心纯化，然后加入外源RNA模板。结果表明，外源dsRNA减少了来自内源性14 kb RNA模板的RNA合成。