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）催化蛋白质合成的易位步骤，在每个连续肽键形成后重新配置核糖体。由于对EF-G催化的复杂易位反应了解甚少，因此分析细菌EF-G的结构和功能非常重要。本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。其中一个被提议的突变区域包括EF-G和其他gtp结合蛋白合成因子的核糖体结合区域。虽然苏氨酸在野生型伸长因子中是不变的，但在这个结构域中已经分离出一个苏氨酸到丝氨酸的突变体，它似乎具有部分功能。有趣的是，将相同的苏氨酸转化为丙氨酸或脯氨酸的突变体在体内的表达会导致生长缓慢的表型。一种高度保守的接近苏氨酸的精氨酸似乎对EF-G功能至关重要。进一步表征这些和其他突变体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有新的认识。活细胞内蛋白质的合成是一个复杂的、多组分的过程，需要消耗大量的能量。细菌蛋白，延伸因子G，或EF-G，在这个过程中是一个重要的参与者。EF-G的作用是准备蛋白质合成装置，以便在合成的蛋白质中添加每一个新的氨基酸。EF-G的作用机制目前所知甚少，尽管对不同生物之间EF-G结构的比较提供了诱人的线索。特别是在EF-G函数中经常发现的结构。本提案的目的是利用生物化学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了解EF-G在蛋白质合成中的作用。了解蛋白质如何合成的细节，以及蛋白质合成是如何与细胞内过程控制和协调的。＊＊＊

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. ***