Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus
Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus
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DOI：
10.1016/s0022-2836(02)01099-9
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发表时间：
2002-12-13
影响因子：
5.6
通讯作者：
Sarnow, P
中科院分区：
文献类型：
--
作者：
Jan, E;Sarnow, P
蟋蟀麻痹病毒（CrPV）是CrPV样病毒家族的成员，其包含单个正链RNA基因组，其编码由短基因间区（IGR）分隔的两个非重叠开放阅读框。CrPV IGR含有指导结构蛋白表达的内部核糖体进入位点（IRES）。与先前描述的IRES不同，IGR IRES通过在不存在起始剂Met-tRNA（i）或任何典型起始因子的情况下从位于核糖体A位点的GCU丙氨酸密码子募集80 S核糖体来起始翻译。在这里，我们已经表明，各种突变，旨在破坏单独的三个假结（PK）结构，并改变高度保守的核苷酸之间的CrPV样病毒，抑制IGR IRES介导的翻译。通过将翻译起始的步骤分为核糖体募集、核糖体定位和核糖体易位，我们发现突变的IRES元件可以分为两类。一类，以PKII和PKIII突变为代表，结合40 S亚基的亲和力显着降低，这表明PKIII和PKII参与了核糖体的初始募集。第二类突变，例如PKI的改变，不影响40 S结合，但改变了定位的核糖体上的IRES，表明PKI参与IRES相关的核糖体的正确定位。这些结果表明，IGR IRES具有独特的假结样结构，其与核糖体进行多次接触，导致起始因子非依赖性募集和核糖体在mRNA上的正确定位。(C)2002爱思唯尔科技有限公司。保留所有权利。
The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80 S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40 S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40 S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA. (C) 2002 Elsevier Science Ltd. All rights reserved.