Ribosomal scanning and initiation codon selection

核糖体扫描和起始密码子选择

• 批准号: 8006444
• 负责人: TATYANA V PESTOVA
• 金额: $ 42.81万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006444
• 关键词: Anticodon; Back; Base Pairing; Binding; Boxing; Cell Proliferation; Cells; Cleaved cell; Codon Nucleotides; Complex; Data; Defect; Development; Developmental Process; Disease; Environment; Erythroid; Eukaryota; Eukaryotic Cell; Event; Family; Fluorescence Resonance Energy Transfer; GTP Binding; Gene Expression; Gene Expression Regulation; Guanosine Triphosphate; Hydrolysis; Hydroxyl Radical; In Vitro; Indium; Individual; Inherited; Initiator Codon; Initiator tRNA; Investigation; Kinetics; Laboratories; Location; Measures; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Morphogenesis; Oncogenes; Open Reading Frames; Peptide Initiation Factors; Physiological; Positioning Attribute; Process; Protein Biosynthesis; Protein Family; Proteins; RNA Helicase; Recycling; Regulation; Regulatory Pathway; Relative (related person); Reporting; Role; Scanning; Staging; Stress; Structure; System; Techniques; Testing; Trans-Activators; Translation Initiation; Translational Derepression; Translations; base; biological adaptation to stress; cell growth; cell transformation; chemical cleavage; crosslink; eukaryotic initiation factor-5B; helicase; in vivo; mRNA capping; member; novel; programs; public health relevance; reconstitution; stop flow technique; thrombocytosis; transcription factor; tumor

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. )

描述（由申请人提供）：该提案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（EIF），并且是多种监管途径的目标。它分为两个阶段：在mRNA的启动密码子及其与60年代核糖体亚基的结合处形成48S启动复合物。首先，43s的预处理复合物包括40S核糖体亚基，EIF2的三元复合物，引发剂tRNA和GTP以及EIFS 3、1和1A附着在限制的5'-透明区域，以EIFS 4A，4A，4B和4F和SCANS的辅助结构的启动，并在限制其二级结构的步骤中，并启动了启动。在启动密码子识别和形成48S复合物，具有已建立的密码子 - 抗原基碱基对，EIF5和EIF5B促进了EIF2结合的GTP的水解，40S子单位的EIF位移以及60s subunit的连接。拟议的研究将基于从单个纯化的翻译成分中的蛋白质合成（启动，伸长，终止和核糖体回收）的体外重构的方法。在AIM 1中，我们将通过确定EIF4E在核糖体启动复合物中的位置来调查EIF4F连接的mRNA进入40S亚基的mRNA结合裂缝的机理方面EIF4G-EIF3-40S相互作用链从核糖体附着到扫描的过渡过程中。 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.我们还建议开发快速动力学技术，以测量扫描的动力学参数，并根据涉及的解旋酶和其他因素来确定它们的差异。 AIM 3将致力于研究各种生理上重要的翻译调节剂的作用机理，这些调节剂与体内研究有关蛋白质合成。在AIM 4中，我们将表征对开始后调节调节的机制，重点是两个过程，优先分类回收的40s亚基回到相同mRNA的5'末端，并在翻译短的开放式阅读框后重新激活。 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将其与发育过程和细胞环境变化相结合的多种监管途径的目标。因此，起始过程中的缺陷会导致严重的遗传疾病，例如遗传性血小板炎和先天性红细胞性促血小板，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中关键事件的分子基础及其对跨性因素的调节，这是开发治疗此类疾病的理性疗法的先决条件。 ）