Studies of Translation Initiation in Bacteria

基因表达的最后一步是翻译，即基于信使RNA(信使RNA)序列合成蛋白质。翻译是由核糖体执行的，核糖体是由核糖体RNA(RRNA)和蛋白质组成的亚细胞机器。近年来，X射线结晶学和冷冻电子显微镜揭示了核糖体的结构及其与各种底物和翻译因子的相互作用。尽管有如此丰富的结构信息，关于核糖体如何在翻译过程中促进特定事件的问题仍然不清楚。这个项目的目标是更好地理解翻译的起始阶段。起始是一个复杂的过程，需要从其他tRNA物种中选择启动子tRNA，并从其他相似或相同的三联体序列中选择起始密码子。在细菌中，这一过程受启动因子IF1、IF2和IF3的动力学控制。在其他生物中，启动从根本上说是相似的，但更复杂，除了普遍保守的IF2(EIF5B)和IF1(EIF1A)外，还涉及10个或更多因素。弗雷德里克实验室已经确定了小(30s)亚单位rRNA的一些突变，这些突变会导致启动过程中的缺陷。本项目将研究大的(50s)亚基和因子IF1、IF3和S1的作用，进一步深入了解控制启动过程的分子机制。更广泛的影响：这个项目的更广泛的影响在于它对教育的高度重视。该项目将为本科生和研究生提供(1)研究培训，(2)为当地高中生(哥伦布公立学校)提供暑期研究机会，以及(3)为俄亥俄州STEM(科学、技术、工程和数学)人才扩展计划的学生提供实验室实习机会，该计划旨在提高STEM专业的成功率。此外，遗传学实验(与项目相关)将被纳入微生物学高级实验室课程。通过进行实际实验而不是实验室练习，这些学生将获得发现的机会，因为他们获得了技术技能。

The final step of gene expression is translation, the synthesis of protein based on the sequence of messenger RNA (mRNA). Translation is performed by ribosomes, sub-cellular machines composed of both ribosomal RNA (rRNA) and protein. In recent years, x-ray crystallography and cryo-electron microscopy have revealed the structure of the ribosome and its interactions with a variety of substrates and translation factors. Despite this wealth of structural information, questions of how the ribosome facilitates specific events during translation remain unclear. The goal of this project is to better understand the initiation phase of translation.Initiation is a complex process that requires selection of the initiator tRNA from other species of tRNA and selection of the start codon from other similar or identical triplet sequences in the mRNA. In bacteria, the process is kinetically controlled by initiation factors IF1, IF2 and IF3. In other organisms, initiation is fundamentally similar but more elaborate, involving 10 or more factors in addition to universally conserved IF2 (eIF5B) and IF1 (eIF1A). The Fredrick Lab has characterized a number of mutations in the rRNA of the small (30S) subunit that cause defects in initiation. This project will investigate the role of the large (50S) subunit and factors IF1, IF3, and S1, providing further insight into the molecular mechanisms that govern the initiation process. BROADER IMPACT: The broader impact of this project lies in its strong educational emphasis. This project will provide (1) research training for undergraduate and graduate students, (2) summer research opportunities for local high school students (Columbus Public Schools), and (3) laboratory internships for students of the Ohio STEM (Science, Technology, Engineering, and Mathematics) Talent Expansion Program, which aims to boost the success rate of STEM majors. In addition, genetic experiments (relevant to the project) will be incorporated into the curriculum of the advanced laboratory course in Microbiology. By conducting actual experiments rather than lab exercises, these students will be given the opportunity for discovery as they gain technical skills.