Structure-Function Analysis of Protein Synthesis Elongation Factor G

小行星9407935 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，在每个连续的肽键形成后重组核糖体。 EF-G催化的复杂易位反应知之甚少，强调了分析细菌EF-G结构和功能的重要性。 本研究的目的是在E. coli EG-G基因的突变，并利用生物化学和遗传学方法分析这些突变的影响。 其中一个建议的诱变区域包括EF-G和其他GTP结合蛋白合成因子的核糖体结合结构域。 已分离出该结构域中的苏氨酸至丝氨酸突变体，其似乎是部分功能性的，即使苏氨酸在野生型延伸因子中是不变的。 有趣的是，其中相同的苏氨酸已被改变为丙氨酸或脯氨酸的突变体的表达导致体内生长缓慢的表型。 一个高度保守的精氨酸接近苏氨酸似乎是必不可少的EF-G功能。 对这些和提案中描述的其他突变体的进一步表征将导致对EF-G结构和功能的更好理解，以及对GTdR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过程，消耗大量的能量。 细菌蛋白，延伸因子G，或EF-G，是这个过程中的重要参与者。 EF-G的作用是准备好蛋白质合成装置，以便将每个新的氨基酸添加到正在合成的蛋白质中。 关于EF-G的作用机制知之甚少，尽管诱人的线索来自不同生物体之间EF-G结构的比较。 特别是，在EF-G功能中总是发现的结构。 该提案的目的是在EF-G蛋白的保守部分中进行离散改变，并使用生物化学方法和细菌遗传学的强大技术的组合来确定这些改变如何影响EF-G功能。 这些研究将有助于更好地了解EF-G如何在蛋白质合成中发挥作用。 了解蛋白质如何合成的细节，以及蛋白质合成如何控制和协调细胞内过程。 ***

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. ***