RNA recognition and cleavage by the SARS coronavirus endoribonuclease

被引:101
作者
Bhardwaj, Kanchan
Sun, Jingchuan
Holzenburg, Andreas
Guarino, Linda A.
Kao, C. Cheng [1 ]
机构
[1] Texas A&M Univ, Dept Biochem & Biophys, College Stn, TX 77843 USA
[2] Texas A&M Univ, Dept Biol, College Stn, TX 77843 USA
[3] Texas A&M Univ, Microscopy & Imaging Ctr, College Stn, TX 77843 USA
[4] Texas A&M Univ, Dept Entomol, College Stn, TX 77843 USA
基金
美国国家科学基金会;
关键词
sARS; coronavirus; endoribonuclease; modified RNAs; hexamer;
D O I
10.1016/j.jmb.2006.06.021
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues.(1-3) How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. 2 We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn21. The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 angstrom structure of Nspl5 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex. (c) 2006 Elsevier Ltd. All rights reserved.
引用
收藏
页码:243 / 256
页数:14
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