Mechanism of EF-G-Dependent Translocation in the Ribosome

蛋白质合成延伸周期的基本步骤之一是tRNA-mRNA复合物在核糖体中的迭代运动，称为易位。这项研究的长期目标是了解trna在大肠杆菌核糖体中从一个位点转移到另一个位点的机制。核糖体由RNA和蛋白质组成。系统发育序列比较、遗传研究和生化实验结果表明核糖体rna在翻译中的功能作用。本提案的具体目标是确定转运所需的a位和p位trna内的功能基团。PI实验室先前的研究表明，含有与p位点结合的全长tRNA的核糖体和与核糖体a位点结合的tRNA的反密码子茎环类似物的核糖体发生易位。这种tRNA的最小模拟物将用于识别必需的核糖2'-羟基和从a位点转移所需的非桥接磷酸氧。PI实验室的其他实验表明，磷酸二酯主链断裂的trna从p位点转移。tRNA较小片段内的特异性修饰可以通过化学合成结合。这为研究转运所需的p位点tRNA中特定官能团的作用提供了一个独特的机会。除了官能团取代研究外，还将使用诸如脚印和化学探测等生化方法来确定核糖体中trna运动所需的相互作用。一旦核糖体的高分辨率晶体结构成为可能，这些研究解决trna和核糖体之间的功能相互作用将变得尤为重要。

0078322JosephOne of the fundamental steps in the elongation cycle of protein synthesis is the iterative movement of the tRNA-mRNA complex in 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. Ribosomes are composed of RNA and proteins. Results from phylogenetic sequence comparison, genetic studies and biochemical experiments indicate a functional role for ribosomal RNAs in translation. The specific goals of this proposal are to identify functional groups within tRNAs in the A-site and P-site that are required for translocation.Previous research in the PI's laboratory has shown that ribosomes containing a full-length tRNA bound to the P-site and an analog of the anticodon stem-loop of tRNA bound to the ribosomal A-site are translocated. This minimal analog of tRNA will be used to identify essential ribose 2'-hydroxyls and non-bridging phosphate oxygens required for translocation from the A-site. Other experiments from the PI's laboratory indicate that tRNAs with a break in the phosphodiester backbone are translocated from the P-site. Specific modifications within the smaller fragments of the tRNA can be incorporated by chemical synthesis. This presents a unique opportunity to study the role of specific functional groups within P-site tRNA that are required for translocation. Biochemical methods such as toeprinting and chemical probing will be used in addition to functional group substitution studies to identify interactions that are required for the movement of the tRNAs in the ribosome. These studies addressing functional interactions between tRNAs and the ribosome will be especially important once high-resolution crystal structures of the ribosome become available.