Structure-Function Analysis of Protein Synthesis Elongation Factor G

9407935 Breitenberger细菌蛋白质合成延伸因子G(EF-G)催化蛋白质合成的转位步骤，在形成每个连续的肽键后重新配置核糖体。人们对EF-G催化的复杂易位反应知之甚少，这突显了分析细菌EF-G结构和功能的重要性。这项研究的目的是在大肠杆菌EG-G基因的特定区域制备定点突变，并使用生化和遗传学方法分析这些突变的影响。建议的突变区域之一包括建议的EF-G核糖体结合区和其他GTP结合蛋白质合成因子。虽然苏氨酸在野生型延伸因子中是不变的，但已分离到该区域的苏氨酸到丝氨酸的突变体，该突变体似乎具有部分功能。有趣的是，在体内表达相同的苏氨酸改变为丙氨酸或脯氨酸的突变体会导致生长缓慢的表型。接近苏氨酸的高度保守的精氨酸似乎是EF-G功能所必需的。对这些突变体和提案中描述的其他突变体的进一步表征将有助于更好地理解EF-G的结构和功能，以及对GTPase超家族其他成员的结构和功能的新见解。活细胞内蛋白质的合成是一个复杂的、多组分的过程，它消耗大量的能量。细菌蛋白质，延伸因子G，或EF-G，是这一过程中必不可少的参与者。EF-G的作用是为蛋白质合成装置做好准备，以便将每一种新的氨基酸添加到正在合成的蛋白质中。虽然诱人的线索来自不同生物体之间的EF-G结构的比较，但人们对EF-G的作用机制知之甚少。特别是EF-G函数中经常出现的结构。这项建议的目的是利用生化方法和强大的细菌遗传学技术相结合，对EF-G蛋白的保守部分进行离散改变，并确定这些变化如何影响EF-G功能。这些研究将有助于更好地理解EF-G如何在蛋白质合成中发挥作用。了解蛋白质是如何合成的，以及蛋白质合成是如何控制和协调细胞内过程的。***

9407935 Breitenberger Bacterial protein synthesis elongation factor G (EF-G) catalyzes the translocation step of protein synthesis, reconfiguring the ribosome after the formation of each successive peptide bond. The complex translocation reaction catalyzed by EF-G is very poorly understood, underscoring the importance of analyzing the structure and function of bacterial EF-G. The objectives of this research are to prepare site-directed mutations in defined regions of the E. coli EG-G gene and to use biochemical and genetic methods to analyze the effects of these mutations. One of the proposed regions for mutagenesis includes the proposed ribosome-binding domain of EF-G and other GTP-binding protein synthesis factors. A threonine to serine mutant in this domain has been isolated, which appears to be partially functional, even though threonine is invariable in wild-type elongation factors. Interestingly, expression of mutants in which the same threonine has been changed to alanine or proline results in a slow-growing phenotype in vivo. A highly conserved arginine close to the threonine seems to essential for EF-G function. Further characterization of these and other mutants described in the proposal will lead to a better understanding of EF-G structure and function, as well as to new insight into the structure and function of other members of the GTPase superfamily. %%% The synthesis of proteins inside living cells is an elaborate, multicomponent process, which consumes large amounts of energy. The bacterial protein, elongation factor G, or EF-G, is an essential participant in this process. The role of EF-G is to ready the protein synthesizing apparatus for the addition of each new amino acid to the protein being synthesized. Very little is known about the mechanism of action of EF-G, although tantalizing clues come from comparisons of EF-G structures between different organisms. In particular, structures which are always found in EF-G function. The objective of this proposal are to make discrete changes in conserved parts of the EF-G protein and to determine how the changes affect EF-G function, using a combination of biochemical methods and the powerful techniques of bacterial genetics. These studies will lead to a better understanding of how EF-G plays its part in protein synthesis. Knowing the details of how proteins are synthesized, and how protein synthesis is controlled and coordinated with intracellular processes. ***