Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes
Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes
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DOI：
10.1093/nar/gkg336
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发表时间：
2003-05-01
影响因子：
14.9
通讯作者：
Nakashima, N
中科院分区：
文献类型：
--
作者：
Nishiyama, T;Yamamoto, H;Nakashima, N
Plautia Stali肠道病毒(PSIV)在基因组间隔区有一个内部核糖体进入位点(IRES)。PSIV IRES用谷氨酰胺而不是普遍的蛋氨酸启动翻译。为了分析IRES介导的启动机制，研究了在没有翻译因子的情况下IRES RNA与盐洗核糖体的结合。在IRES内的三个假结(PK I、PK II和PK III)中，PK III对核糖体结合最重要。化学足迹分析表明，结构域2的两个茎环结构的环部分在相关病毒中高度保守，受到40s核糖体的保护，而不受60s核糖体的保护。由于PK III靠近这两个环，这些结构元件被认为对40S亚基的结合是重要的。竞争结合分析表明，IRES RNA不与tRNA(Phe)或其反密码子茎环(ASL)片段预先孵育的聚(U)编程核糖体结合。然而，结构域3缺失的IRES与与ASL预先孵育的编程核糖体结合，表明结构域1和2在IRES与40S亚基结合中起作用，结构域3位于核糖体解码位置。
Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem-loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA(Phe) or its anticodon stem- loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.