CAREER: Molecular Interactions and Nucleotide Exchange Mechanism of Translation Factor EF1B Alpha

职业：翻译因子 EF1B Alpha 的分子相互作用和核苷酸交换机制

• 批准号: 9983565
• 负责人: Terri Kinzy
• 金额: $ 49.91万
• 依托单位: Rutgers, The State University of New Jersey-RBHS-Robert Wood
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9983565&HistoricalAwards=false
• 关键词: CAREER; Molecular; Interactions; Nucleotide; Exchange

9983565KinzyG-proteins are a broad class of proteins whose activity is regulated by the phosphorylation state of their bound nucleotide. When bound to the tri-phosphate form of guanosine (GTP), they are active in a multitude of biological processes such as protein synthesis. Stimulation of the G-protein's hydrolytic activity by another factor, a GTPase activating protein or GAP, converts the G-protein to the inactive diphosphate (GDP)-bound form. Another set of regulatory proteins termed guanine nucleotide exchange factors (GEFs) control the nucleotide bound state of G-proteins by facilitating the conversion back to the active GTP bound form. The translation Elongation Factor 1Balpha (eEF1Balpha, formerly EF-1beta) of the Baker's yeast Saccharomyces cerevisiae is a GEF for the elongation step of protein synthesis. As a small, essential protein, it serves as an excellent system to utilize biochemical and genetic approaches to the study of the mechanism of guanine nucleotide exchange and the regulation of the elongation phase of protein synthesis. eEF1Balpha functions on the G-protein eEF1A (formerly EF-1alpha) which is the homolog of the first G-protein for which the crystal structure was know, bacterial EF-Tu. eEF1A is active to bind and deliver activated amino acids attached to transfer RNAs to the ribosome only when in the GTP-bound form. The GDP to GTP exchange catalyzed by eEF1Balpha is essential to maintain a pool of active eEF1A and is likely the rate-limiting step of translation elongation. The PI's large bank of mutant forms of yeast eEF1Balpha has demonstrated the critical role of this protein in efficient and accurate protein synthesis in cells. The mechanism and regulation of the guanine nucleotide exchange process will be determined utilizing our available as well as new structurally based point mutations. Purified factors and in vitro assays for protein synthesis will identify the critical functional motifs of eEF1Balpha and dissect the interaction between this GEF and its G-protein eEF1A. The cellular growth and translation defects of yeast strain with a mutant form of eEF1Balpha allow us to utilize genetic approaches to identify factors that may regulate the GEF activity of this protein as well as the translation elongation process in general. Thus, the yeast S. cerevisiae is an eukaryotic system that permits simultaneous analysis of the role of eEF1Balpha in the translation elongation cycle in vivo and in vitro. The educational goals of this proposal utilize both existing minority enhancement programs and the work study program for undergraduates at Rutgers University in our research program to understand the role of eEF1Balpha in gene expression. The University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School (formerly Rutgers Medical School) has a tradition of supporting undergraduate research and minority enhancement programs. The work study placement program augments established programs with a system to identify economically disadvantaged students for laboratory research. The P.I., as the director of the UMDNJ RWJMS DNA Sequencing core facility, is also involved in the integration of state of the art molecular biology techniques into high school student and teacher programs and undergraduate courses at Rutgers University.

9983565 KinzyG蛋白是一类广泛的蛋白质，其活性受其结合核苷酸的磷酸化状态调节。 当与鸟苷的三磷酸形式（GTP）结合时，它们在许多生物过程中具有活性，例如蛋白质合成。 通过另一种因子，GT3活化蛋白或GAP刺激G蛋白的水解活性，将G蛋白转化为无活性的二磷酸（GDP）结合形式。 另一组称为鸟嘌呤核苷酸交换因子（GEF）的调节蛋白通过促进转化回活性GTP结合形式来控制G蛋白的核苷酸结合状态。 面包酵母酿酒酵母的翻译延伸因子1B α（eEF 1B α，以前称为EF-1 β）是蛋白质合成延伸步骤的GEF。 作为一个小的，必需的蛋白质，它作为一个很好的系统，利用生物化学和遗传学的方法来研究鸟嘌呤核苷酸交换的机制和蛋白质合成的延伸阶段的调节。 eEF 1B α作用于G蛋白eEF 1A（以前称为EF-1 α），它是已知晶体结构的第一种G蛋白的同系物，即细菌EF-Tu。 eEF 1A只有在GTP结合形式时才有活性结合并递送与转移RNA连接的活化氨基酸至核糖体。 由eEF 1B α催化的GDP到GTP的交换对于维持活性eEF 1A池是必不可少的，并且可能是翻译延伸的限速步骤。PI的酵母eEF 1B α突变形式的大型银行已经证明了这种蛋白质在细胞中有效和准确的蛋白质合成中的关键作用。 鸟嘌呤核苷酸交换过程的机制和调节将利用我们现有的以及新的基于结构的点突变来确定。 纯化的因子和蛋白质合成的体外测定将鉴定eEF 1B α的关键功能基序，并分析该GEF与其G蛋白eEF 1A之间的相互作用。 具有eEF 1B α突变形式的酵母菌株的细胞生长和翻译缺陷使我们能够利用遗传方法来鉴定可能调节该蛋白的GEF活性以及一般翻译延伸过程的因素。 因此，酵母S.酿酒酵母是一个真核系统，允许在体内和体外同时分析eEF 1B α在翻译延长周期中的作用。 本提案的教育目标利用现有的少数民族增强计划和罗格斯大学本科生的工作学习计划，以了解eEF 1B α在基因表达中的作用。 新泽西医学和牙科大学罗伯特·伍德约翰逊医学院（原罗格斯医学院）有支持本科生研究和少数民族增强计划的传统。 勤工俭学安置计划增加了既定的计划与系统，以确定经济困难的学生进行实验室研究。 私家侦探，作为UMDNJ RWJMS DNA测序核心设施的负责人，还参与了将最先进的分子生物学技术整合到罗格斯大学的高中学生和教师计划以及本科课程中。