Regulation of eukaryotic translation initiation

描述(由申请人提供)： 翻译启动是一个多步骤的过程，涉及大量的真核启动因子(ELF)，最终协调Met-tRNAiMet碱基与80s核糖体上mRNA的Aug起始密码子的组装。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、细胞生长和细胞凋亡中起着关键作用，而它的解除调控会导致细胞周期控制的丧失和细胞的恶性转化。在所有的启动因子中，elF3特别耐人寻味，因为它刺激了通路中的多个步骤，如Met-tRNAiMet和mRNA与40S核糖体的结合。具体的假设是，酵母elF3促进40S与先前在AUG识别过程中涉及的ELF1、2和5的结合，从而共同调节40S核糖体扫描mRNA的5‘UTR直到遇到起始AUG密码子的过程。我们基于这样的假设：A)酵母elF3与elFS 1，5和Met-tRNAiMet-elF2-GTP三元复合体(TC)形成多因子复合体(MFC)，可以在没有核糖体的情况下存在；B)我们发现elF3与40S核糖体之间的几个潜在接触似乎对MFC传递到核糖体很重要；以及C)elF3 NIP1亚基上的簇状丙氨酸突变似乎影响43S复合体的形成和AUG识别的严密性。要解决这个假设，请执行以下操作： 1)我们将利用酵母遗传学和生物化学技术，例如体外结合分析、体内亲和层析、甲醛交联和蔗糖梯度沉淀法分离提取物，突变elF3-40S结合位点并分析它们的生理意义；以及2)尝试确定与40S的额外MFC接触。3)我们将对elF3的NIP1亚单位的选定片段进行定点突变，该片段介导与elf1、2和5的相互作用，然后对突变表型进行彻底分析，表明Aug选择的严格程度放松，TC招募的缺陷持续到40s。

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.