Ribosomal scanning and initiation codon selection

 描述(申请人提供)：起始是真核蛋白质合成过程中最复杂的阶段，也是多种调控途径的靶标。翻译装置及其调控的缺陷会导致各种破坏性的疾病，因此启动越来越成为化疗干预的焦点。因此，了解引发反应的分子机制是一个重要的优先事项，但这一过程中几个步骤的细节仍然知之甚少。概括地说，真核细胞的启动如下所示。Met-tRNAiMet、eIF2和GTP形成一个三元复合体，与EIFS3、1和1A一起与40S亚基结合，形成43S预引发复合体。43S复合体连接到mRNA的帽-近端区域，涉及到EIFS4A、4B和4F解开其二级结构，然后扫描到起始密码子，在那里它们形成具有已建立的密码子-反密码子碱基配对的48S起始复合体。扫描结构化的mRNA严格需要DHX29，这是一种直接与40S亚基结合的DExH-box蛋白。EIFS5和5B促进60S亚基随后连接到48S复合体，形成具有伸长能力的80S核糖体。我们对整个翻译过程的体外重建的发展，以及最近这些方法与我们的合作者J.Frank(HHMI，哥伦比亚大学)在冷冻电子显微镜方面所取得的进展的集成，为弥合理解哺乳动物启动机制方面的几个关键空白提供了独特的机会。该提案的目标1-3将解决规范启动过程第一阶段中尚未解决的关键方面：Met-tRNAiMet的核糖体招募、43S复合体与mRNA的结合和核糖体扫描。目的1将重点介绍eIF2促进Met-tRNAiMet与40S亚基结合的机制，特别是(I)eIF2亚基在这一过程中的作用，最近发现eIF2亚基是介导eIF2与43S复合体中40S亚基唯一稳定的接触的；(Ii)ABC50刺激eIF2活性的机制。在目标2中，我们将继续进一步研究eIF4E结合的封端mRNAs在43S复合体附着过程中进入40S亚单位的mRNA结合裂缝的机制，并将表征eIF4B在这一过程中的作用。目的3将致力于确定DHX29促进核糖体扫描的机制，特别是区分DHX29通过诱导40S亚基的构象变化而直接解开mR NA和间接地发挥DHX29的S作用模式。最后一个目标4将涉及非Aug密码子的非规范启动模式：(I)用Leu-tRNALeu启动CUG密码子，这有可能成为一种允许在eIF2磷酸化条件下进行翻译的调节机制，以及(Ii)在重复相关的非Aug(RAN)翻译过程中，在非Aug三联体上启动，这发生在mRNAs的非编码区的三联体/六联体扩展重复上，这些基因是许多严重的神经退行性疾病的基因转录而来的。

 DESCRIPTION (provided by applicant): Initiation is the most complex stage of eukaryotic protein synthesis, and is a target for multiple regulatory pathways. Defects in the translation apparatus and its regulation cause a variety of devastating diseases, and initiation is therefore increasingly becoming a focus for chemo-therapeutic intervention. Knowledge of molecular mechanisms underlying initiation is thus an important priority, but details of several steps in thi process remain poorly understood. In outline, eukaryotic initiation occurs as follows. Met-tRNAiMet, eIF2 and GTP form a ternary complex, which together with eIFs 3, 1, and 1A bind to 40S subunits, yielding a 43S preinitiation complex. 43S complexes attach to the cap-proximal region of mRNA in a step that involves unwinding of its secondary structure by eIFs 4A, 4B and 4F, and then scan to the initiation codon, where they form 48S initiation complexes with established codon-anticodon base-pairing. Scanning on structured mRNAs strictly requires DHX29, a DExH-box protein that binds directly to 40S subunits. Subsequent joining of 60S subunits to 48S complexes and formation of elongation-competent 80S ribosomes is promoted by eIFs 5 and 5B. Our development of in vitro reconstitution of the entire translation process and recent integration of these approaches with advances in cryo-electron microscopy achieved by our collaborator J. Frank (HHMI, Columbia University) gives unique opportunities to close several critical gaps in understanding of the mechanism of mammalian initiation. Aims 1-3 of this proposal will address the key unresolved aspects of the first stages in the canonical initiation process: ribosomal recruitment of Met-tRNAiMet, attachment of 43S complexes to mRNA and ribosomal scanning. Aim 1 will focus on the mechanism by which eIF2 promotes binding of Met-tRNAiMet to the 40S subunit, in particular, (i) the role in this process of the eIF2 subunit, which was recently found to mediate the only stable contact of eIF2 with the 40S subunit in 43S complexes, and (ii) the mechanism by which the activity of eIF2 is stimulated by ABC50. In Aim 2, we will continue to further investigate the mechanistic aspects of entry of eIF4E-bound capped mRNAs into the mRNA-binding cleft of the 40S subunit during attachment of 43S complexes, and will characterize the role of eIF4B in this process. Aim 3 will be devoted to determination of the mechanism by which DHX29 promotes ribosomal scanning, in particular, to discrimination between direct unwinding of mRNA by DHX29 and an indirect mode of DHX29's action by inducing conformational changes in the 40S subunit. The last Aim 4 will concern non-canonical modes of initiation on non-AUG codons: (i) initiation on CUG codons with Leu-tRNALeu, which has the potential to be a regulatory mechanism allowing translation in conditions of eIF2 phosphorylation, and (ii) initiation on non-AUG triplets during repeat-associated non-AUG (RAN) translation, which occurs on triplet/sextet expansion repeats in noncoding regions of mRNAs transcribed from genes that are responsible for many severe neurodegenerative diseases.