Regulation of eukaryotic translation initiation

真核翻译起始的调控

基本信息

批准号：
7254870
负责人：
Leos Valasek
金额：
$ 5.12万
依托单位：
INSTITUTE OF MICROBIOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7254870
关键词：
Address Affect Affinity Chromatography Alanine Animal Model Apoptosis Base Pairing Binding Binding Sites Biochemistry Biological Assay C-terminal Cell Cycle Progression Cell Cycle Regulation Cell Extracts Charge Chromatography Cluster Analysis Codon Nucleotides Collaborations Complex DNA Data Defect Development Ensure Eukaryota Eukaryotic Cell Eukaryotic Initiation Factors Foundations Fractionation Free Ribosome GTPase-Activating Proteins Gene Expression Genetic Goals Helix (Snails)In Vitro Individual Initiator Codon Malignant - descriptor Malignant Neoplasms Maps Mediating Messenger RNA Monitor Mutagenesis Mutate Mutation N-terminal National Institute of Child Health and Human Development Northern Blotting Numbers Pathway interactions Peptide Initiation Factors Phase Phenotype Physiological Play Positioning Attribute Principal Investigator Process Protein Binding Protein Biosynthesis Protein Subunits Proteins RNA, Transfer, Met RNA-Binding Proteins Rate Reaction Regulation Research Research Personnel Ribosomal Proteins Ribosomal RNA Ribosomes Role Saccharomyces cerevisiae Saccharomycetales Scanning Sedimentation process Side Site Site-Directed Mutagenesis Solvents Sucrose Techniques Temperature Testing Translation Initiation Translational Regulation Translations Untranslated Regions Vertebral column Yeasts base cell growth cell transformation chelation crosslink ear helix genetic analysis in vivo insight interest malignant phenotype mutant prevent programs prospective response scaffold tumorigenesis yeast genetics yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Translation initiation is a multiple-step process involving a large number of eukaryotic initiation factors (elFs) that ultimately orchestrate assembly of the Met-tRNAiMet base-paired with the AUG start codon of mRNA on the 80S ribosome. Our long-term goal is to elucidate the regulation of this process as it plays a critical role in development, differentiation, cell cycle progression, cell growth, and apoptosis and its deregulation results in a loss of cell cycle control and malignant transformation of cells. Of all the initiation factors, elF3 is particularly intriguing because it stimulates multiple steps in the pathway such as binding of Met-tRNAiMet and mRNA to the 40S ribosome. The specific hypothesis is that yeast elF3 promotes 40S-binding of elFs 1, 2, and 5, that were previously implicated in the AUG recognition process, and thus co-regulates the process during which the 40S ribosome scans the 5' UTR of mRNA until it encounters the start AUG codon. We base that hypothesis on the observations that A) yeast elF3 forms a multifactor complex (MFC) with elFs 1, 5 and Met-tRNAiMet-elF2-GTP ternary complex (TC) that can exist free of ribosomes, B) several potential contacts that we found between elF3 and the 40S ribosome seem to be important for the MFC delivery to the ribosome, and C) clustered-alanine mutations in NIP1 subunit of elF3 appear to influence 43S complex formation and stringency of AUG recognition. To address this hypothesis: 1) We will mutate the elF3-40S binding sites and analyze their physiological importance using techniques of yeast genetics and biochemistry, e.g. in vitro binding assays, in vivo affinity chromatography, HCHO cross-linking and fractionation of extracts by sucrose gradients sedimentation; and 2) attempt to identify additional MFC contacts with 40S. 3) We will conduct a site-directed mutagenesis of the selected segments of the NIP1 subunit of elF3 that mediates interactions with elFs 1, 2, and 5 followed by thorough analysis of mutant phenotypes indicating relaxed stringency of AUG selection and defects in TC recruitment to 40S.

描述（由申请人提供）：翻译起始是一个多步骤过程，涉及大量真核起始因子 (elF)，最终协调与 80S 核糖体上 mRNA 的 AUG 起始密码子配对的 Met-tRNAiMet 的组装。我们的长期目标是阐明这一过程的调节，因为它在发育、分化、细胞周期进展、细胞生长和细胞凋亡中发挥着关键作用，其失调会导致细胞周期控制丧失和细胞恶性转化。在所有启动因子中，elF3 特别令人感兴趣，因为它刺激途径中的多个步骤，例如 Met-tRNAiMet 和 mRNA 与 40S 核糖体的结合。具体假设是，酵母elF3促进之前参与AUG识别过程的elFs 1、2和5的40S结合，从而共同调节40S核糖体扫描mRNA 5'UTR的过程，直到它遇到起始密码子 AUG。我们的假设基于以下观察结果：A) 酵母 elF3 与 eFs 1、5 和 Met-tRNAiMet-elF2-GTP 三元复合物 (TC) 形成多因子复合物 (MFC)，该复合物可以不存在核糖体，B) 几种潜在的接触我们发现 elF3 和 40S 核糖体之间似乎对于 MFC 向核糖体的递送很重要，并且 C) 丙氨酸簇突变eF3 的 NIP1 亚基中的 43S 复合物形成和 AUG 识别的严格性似乎受到影响。为了解决这个假设： 1）我们将使用酵母遗传学和生物化学技术对elF3-40S结合位点进行突变并分析其生理重要性，例如体外结合测定、体内亲和层析、HCHO 交联以及通过蔗糖梯度沉淀对提取物进行分级分离； 2) 尝试识别与 40S 的其他 MFC 接触。 3）我们将对elF3的NIP1亚基的选定片段进行定点诱变，介导与elFs 1、2和5的相互作用，然后对突变表型进行彻底分析，表明AUG选择的宽松严格性和TC招募中的缺陷40秒。