Mechanism Of Ef-G-Dependent Translocation In The Ribosome

翻译过程中的一个基本步骤是核糖体内mRNA-tRNA复合物的精确和协调运动，称为易位。本研究的长期目标是了解核糖体易位的分子机制。PI实验室先前的研究表明，A和P位点trna中的特定核糖2'-羟基和非桥接磷酸氧原子对易位很重要。当前项目的目标是确定mRNA中易位所需的2'-羟基和非桥接磷酸氧原子。此外，目前的项目将直接研究16S rRNA中普遍保守的碱基在易位过程中与mRNA-tRNA复合物相互作用的作用。该研究将使用基于荧光的稳态前动力学分析、关键残基的定点诱变和生化分析来研究易位的机制。这些实验将提供关于核糖体结构动力学的信息，这些信息是x射线晶体学无法轻易获得的。细菌核糖体是几种抗生素灭活的目标。抗生素如红霉素、维霉素、硫链霉素和大观霉素能特异性抑制易位。了解翻译的机制将为开发新型抗生素提供新的见解。该项目还通过整合研究和教学来满足重要的教育需求。这项研究将促进先进生物物理方法的教学和培训。少数民族本科生、研究生和博士后将在实验室获得宝贵的研究经验。

One of the fundamental steps during translation is the precise and coordinated movement of the mRNA-tRNA complex within the ribosome, called translocation. The long-term goal of this research is to understand the molecular mechanism of translocation by the ribosome. Previous studies in the PI's laboratory showed that specific ribose 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the A and P site tRNAs are important for translocation. The goal of the current project is to identify 2'-hydroxyl groups and non-bridging phosphate oxygen atoms in the mRNA that are required for translocation. Additionally, the current project will directly examine the role of the universally conserved bases in 16S rRNA that interacts with the mRNA-tRNA complex in translocation. The research will use fluorescence-based pre-steady state kinetic analysis, site-directed mutagenesis of critical residues, and biochemical assays to study the mechanism of translocation. These experiments will provide information about the dynamics of ribosome structure that cannot be easily acquired by X-ray crystallography. 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 mechanism of translation will provide insights for developing novel antibiotics. This project also serves an important educational need by integrating research and teaching. The research will promote teaching and training in advanced biophysical methods. Several undergraduate minority students, graduate students and postdoctoral students will obtain invaluable research experience in the laboratory.