Mechanism of EF-G-Dependent Translocation In The Ribosome

核糖体中 EF-G 依赖性易位机制

• 批准号: 0315780
• 负责人: Simpson Joseph
• 金额: $ 36万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0315780&HistoricalAwards=false
• 关键词: Mechanism; EF; Dependent; Translocation; Ribosome

Ribosomes carry out the complex task of protein synthesis in all organisms. A fundamental step during protein biosynthesis is the precise and coordinated movement of the tRNA-mRNA complex within the ribosome, called translocation. The long-term objective of this research is to understand the mechanism of translocation of tRNAs from one site to the next within the E.coli ribosome. The specific goal of this project is to determine the role of two well-characterized base pairs between 23S rRNA and P and A site tRNAs in translocation. The first base pair is between the universally conserved bases C74 of P site tRNA and G2252 of 23S rRNA. The second base pair interaction is between universally conserved bases C75 of A site tRNA and G2553 of 23S rRNA. The effect of disrupting these base pairs on the kinetics of translocation will be examined using a rapid kinetic method recently developed in the PI's laboratory. In addition,methods such as site-directed mutagenesis, toeprinting, and chemical probing will be used to characterize the role of these universally conserved base pairs in translocation. Bacterial ribosomes are the target for inactivation by several classes of antibiotics. Antibiotics such as erythromycin, viomycin, thiostrepton, and spectinomycin specifically inhibit translocation. Understanding the functional role of specific interactions within the ribosome is critical for elucidating the molecular mechanism of translation and will provide insights for developing novel antibiotics. This research also serves an important educational need by integrating research and education. Several undergraduate minority students will obtain invaluable research experience in the laboratory.

核糖体在所有生物体中执行蛋白质合成的复杂任务。 蛋白质生物合成过程中的一个基本步骤是核糖体内 tRNA-mRNA 复合物的精确协调运动，称为易位。 这项研究的长期目标是了解大肠杆菌核糖体中 tRNA 从一个位点易位到下一个位点的机制。 该项目的具体目标是确定 23S rRNA 与 P 和 A 位点 tRNA 之间两个已明确表征的碱基对在易位中的作用。 第一个碱基对位于 P 位点 tRNA 的普遍保守碱基 C74 和 23S rRNA 的 G2252 之间。 第二个碱基对相互作用发生在 A 位点 tRNA 的普遍保守碱基 C75 和 23S rRNA 的 G2553 之间。 破坏这些碱基对对易位动力学的影响将使用 PI 实验室最近开发的快速动力学方法进行检查。 此外，定点诱变、脚趾印迹和化学探测等方法将用于表征这些普遍保守的碱基对在易位中的作用。 细菌核糖体是几类抗生素灭活的目标。 红霉素、紫霉素、硫链丝菌素和壮观霉素等抗生素可特异性抑制易位。 了解核糖体内特定相互作用的功能作用对于阐明翻译的分子机制至关重要，并将为开发新型抗生素提供见解。 这项研究还通过整合研究和教育来满足重要的教育需求。 几名本科少数民族学生将在实验室获得宝贵的研究经验。