Ribosomal scanning and initiation codon selection

描述（申请人提供）：本次申报的重点是哺乳动物翻译起始的机制，该机制至少需要9个起始因子（eIFs），是多个调控途径的靶点。它发生在两个阶段：在mRNA的起始密码子处形成48 S起始复合物，并与60 S核糖体亚基连接。首先，包含40 S核糖体亚基、eIF 2、起始tRNA和GTP的三元复合物以及eIFs 3、1和1A的43 S前起始复合物在涉及通过eIFs 4A、4 B和4F解旋其二级结构的步骤中附着于mRNA的加帽5 '近端区域，然后扫描至起始密码子。在起始密码子识别和形成具有已建立的密码子-反密码子碱基配对的48 S复合物后，eIF 5和eIF 5 B促进eIF 2结合的GTP的水解、eIFs从40 S亚基的置换和60 S亚基的连接。拟定研究将基于从单个纯化翻译组分体外重建蛋白质合成所有阶段（起始、延伸、终止和核糖体再循环）的方法。在目标1中，我们将通过确定eIF 4 E在核糖体起始复合物中的位置，鉴定加帽mRNA中AUG密码子可以与起始tRNA有效相互作用的第一个位置，以及在从核糖体附着到扫描的过渡期间，追踪cap-eIF 4 E-eIF 4G-eIF 3 - 40 S相互作用链的命运。在目标2中，我们提出研究目前涉及启动的DEAD/DExH盒蛋白（例如eIF 4A，Ded 1，DHX 29等）的网络。通过表征它们在起始的不同阶段（核糖体附着和扫描）的相对个体活性以及它们在通过稳定的mRNA二级结构促进核糖体扫描中的潜在协同作用。我们还建议开发快速动力学技术来测量扫描的动力学参数，并确定它们如何根据所涉及的解旋酶和其他因素而有所不同。目的3将致力于研究各种生理上重要的翻译调节剂的作用机制，这些调节剂通过体内研究与蛋白质合成有关。在目标4中，我们将描述启动的后循环调节机制，重点是两个过程，再循环的40 S亚基优先分流回相同mRNA的5 '端，以及短开放阅读框翻译后的重新启动。 公共卫生相关性：蛋白质合成在细胞代谢中至关重要，其复杂的起始阶段是多种调控途径的目标，这些途径将其与发育过程和细胞环境的变化相结合。因此，起始过程中的缺陷可引起严重的遗传性疾病，例如遗传性血小板增多症和先天性红细胞发育不全，以及异常的细胞生长和增殖，例如在肿瘤中。这些研究将确定翻译起始关键事件的分子基础，以及反式作用因子对其的调节，这是开发治疗此类疾病的合理疗法的先决条件。)

DESCRIPTION (provided by applicant): The focus of this proposal is the mechanism of mammalian translation initiation, which requires at least 9 initiation factors (eIFs), and is a target for multiple regulatory pathways. It occurs in two stages: formation of a 48S initiation complex at the initiation codon of mRNA and its joining with a 60S ribosomal subunit. First, 43S preinitiation complex comprising a 40S ribosomal subunit, a ternary complex of eIF2, initiator tRNA and GTP, and eIFs 3, 1 and 1A attaches to the capped 5'-proximal region of mRNA in a step that involves unwinding of its secondary structure by eIFs 4A, 4B and 4F, and then scans to the initiation codon. After initiation codon recognition and formation of the 48S complex with established codon-anticodon base-pairing, eIF5 and eIF5B promote hydrolysis of eIF2-bound GTP, displacement of eIFs from the 40S subunit and joining of a 60S subunit. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosomal recycling) from individual purified translational components. In Aim 1, we will investigate the mechanistic aspects of entry of eIF4F-bound capped mRNAs into the mRNA-binding cleft of the 40S subunit during attachment of 43S complexes by determining the position of eIF4E in ribosomal initiation complexes, identifying the first position in a capped mRNA at which an AUG codon can interact productively with initiator tRNA, and following the fate of the cap-eIF4E- eIF4G-eIF3-40S chain of interactions during the transition from ribosomal attachment to scanning. In Aim 2, we propose to investigate the network of DEAD/DExH-box proteins that have currently been implicated in initiation (e.g. eIF4A, Ded1, DHX29 etc.) by characterizing their relative individual activities at distinct stages of initiation (ribosomal attachment and scanning) and their potential synergy in promoting ribosomal scanning through stable mRNA secondary structures. We also propose to develop fast kinetics techniques to measure kinetic parameters of scanning and to determine how they differ depending on the helicases and other factors involved. Aim 3 will be devoted to investigation of the mechanism of action of various physiologically important translation regulators that have been implicated in protein synthesis by studies in vivo. In Aim 4, we will characterize mechanisms of post-recycling regulation of initiation, focusing on two processes, preferential shunting of recycled 40S subunits back to the 5'-end of the same mRNA, and reinitiation after translation of short open reading frames. PUBLIC HEALTH RELEVANCE: Protein synthesis is of central importance in cell metabolism, and its complex initiation stage is a target for multiple regulatory pathways that integrate it with developmental processes and with changes in the cellular environment. Accordingly, defects in the initiation process can cause severe inherited diseases such as hereditary thrombocythemia and congenital erythroid aplasia, and aberrant cell growth and proliferation, for example in tumors. These studies will determine the molecular basis for key events in translation initiation, and its regulation by trans-acting factors, which is a prerequisite for the development of rational therapies to treat such diseases. )