A novel insight into the mechanism of mammalian selenoprotein synthesis.
A novel insight into the mechanism of mammalian selenoprotein synthesis.
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DOI：
10.1261/rna.036871.112
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发表时间：
2013-08
期刊：
影响因子：
--
通讯作者：
Karpova G
中科院分区：
文献类型：
--
作者：
Kossinova O;Malygin A;Krol A;Karpova G
对硒半胱氨酸插入UGA密码子很重要的成分已经知道一段时间了。通过使用交叉链接的方法，本文对这些组件与翻译机制的交互机制提供了新的见解。氨基酸硒半胱氨酸是由UGA编码的，通常是一个终止密码子，因此需要一种特殊的机制才能将其整合到硒蛋白中。该机制包括tRNASec，一个被称为SElenoCysteine插入序列(SECIS)的3‘-UTRmRNA茎环，SECIS是将UGA重新编码为SEC密码子所必需的，SECIS结合蛋白2(SBP2)，以及其他蛋白质。关于分子机制，特别是SECIS和SBP2何时、何地和如何与核糖体接触，人们知之甚少。其他人之前的工作使用分离的SECIS RNA来解决这个问题。在这里，我们开发了一种新的方法，使用工程设计的最小硒蛋白mRNAs，其中包含用光反应基团衍生的SECIS元件。通过兔网织红细胞裂解液的交联实验，可以获得关于SBP2和SECIS在翻译的各个步骤与翻译机器的组件接触的新信息。特别是，我们发现SBP2只在48S预引发和80S预易位复合体中与SECIS结合。在SEC-tRNASec与A位点结合但转肽被阻断的复合体中，SBP2与核糖体结合，可能还与SECIS元件结合，SECIS与60S核糖体亚基灵活接触，涉及几种核糖体蛋白。总之，我们的发现拓宽了我们对哺乳动物中硒半胱氨酸掺入的独特机制的理解。
The components which are important for selenocysteine insertion into UGA codons have been known for some time. Using a cross-linking approach, this paper provides new insight into the mechanism by which these components interact with the translation machinery. The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNASec, a 3′-UTR mRNA stem–loop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNASec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals.
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