Regulation of eukaryotic translation initiation

真核翻译起始的调控

• 批准号: 7388870
• 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
• 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/7388870
• 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

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）： 翻译起始是涉及大量真核起始因子（eIF）的多步骤过程，其最终协调与80 S核糖体上mRNA的AUG起始密码子碱基配对的Met-tRNAiMet的组装。我们的长期目标是阐明这一过程的调控，因为它在发育、分化、细胞周期进展、细胞生长和凋亡中起着关键作用，而其失调导致细胞周期控制丧失和细胞恶性转化。在所有的起始因子中，eIF 3是特别有趣的，因为它刺激途径中的多个步骤，例如Met-tRNAiMet和mRNA与40 S核糖体的结合。具体的假设是酵母eIF 3促进先前参与AUG识别过程的eIF 1、2和5的40 S-结合，并因此共调节40 S核糖体扫描mRNA的5' UTR直到其遇到起始AUG密码子的过程。我们将该假设基于以下观察：A）酵母eIF 3与eIF 1、5和Met-tRNAiMet-eIF 2-GTP三元复合物（TC）形成多因子复合物（MFC），其可以不含核糖体存在，B）我们发现eIF 3和40 S核糖体之间的几种潜在接触似乎对于MFC递送至核糖体是重要的，和C）eIF 3的NIP 1亚基中的成簇丙氨酸突变似乎影响43 S复合物的形成和AUG识别的严格性。为了解决这一假设： 1)我们将突变eIF 3 - 40 S结合位点，并使用酵母遗传学和生物化学技术分析其生理重要性，例如体外结合测定、体内亲和色谱、HCHO交联和通过蔗糖梯度沉降分级分离提取物;和2）尝试鉴定与40 S的额外MFC接触。3)我们将对介导与eIFs 1、2和5相互作用的eIF 3的NIP 1亚基的选定片段进行定点诱变，然后对突变体表型进行彻底分析，表明AUG选择的严格性放松，TC募集到40 S的缺陷。