Ribosomal scanning and initiation codon selection

描述（由申请人提供）：本提案的重点是哺乳动物翻译启动机制，该机制需要至少9个启动因子（eif），并且是多种调控途径的靶标。它发生在两个阶段：mRNA起始密码子的48S起始复合物的形成及其与60S核糖体亚基的结合。首先，由40S核糖体亚基、eIF2、启动物tRNA和GTP以及eIFs 3、1和1A组成的43S起始前复合物附着在mRNA的5'近端区域，这一步涉及到eIFs 4A、4B和4F对其二级结构的解绕，然后扫描到起始密码子。eIF5和eIF5B在识别起始密码子并与已建立的密码子-反密码子碱基配对形成48S复合物后，促进eif2结合的GTP水解，将eIFs从40S亚基中移出并加入60S亚基。拟议的研究将基于从单个纯化的翻译组分中体外重建蛋白质合成的所有阶段（起始，延伸，终止和核糖体再循环）的方法。在Aim 1中，我们将通过确定eIF4E在核糖体起始复合物中的位置，研究eif4f结合的带帽mRNA在43S复合物附着过程中进入40S亚基mRNA结合间隙的机制，确定在带帽mRNA中AUG密码子能够与启动物tRNA有效相互作用的第一个位置。并跟踪cap-eIF4E- eIF4G-eIF3-40S相互作用链在从核糖体附着到扫描的转变过程中的命运。在Aim 2中，我们建议通过表征它们在起始（核糖体附着和扫描）的不同阶段（核糖体附着和扫描）的相对个体活性，以及它们通过稳定的mRNA二级结构促进核糖体扫描的潜在协同作用，来研究目前与起始（例如eIF4A， Ded1， DHX29等）有关的DEAD/DExH-box蛋白网络。我们还建议发展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解旋酶和其他因素而不同。目标3将致力于研究各种生理上重要的翻译调节因子的作用机制，这些调节因子在体内研究中涉及蛋白质合成。在Aim 4中，我们将描述再生后启动调控的机制，重点关注两个过程，即再生40S亚基优先转回同一mRNA的5'端，以及翻译短开放阅读框后重新启动。

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. )