COLLABORATIVE RESEARCH: The Role of Quality Control in Microbial Translation

智力价值：为了正常运作，细胞必须在处理遗传信息转移的所有过程中保持一定程度的保真度。翻译是通过读取核酸模板序列中编码的遗传信息来使蛋白质具有相应的氨基酸序列的过程。翻译的准确性取决于两个关键过程的准确性：氨基酰基-tRNA合成酶(AARS)合成同源氨基酸/tRNA对，以及核糖体选择正确的氨基酰基-tRNA以匹配mRNA模板上的下一个密码子。AARs通过将tRNA与相应的氨基酸配对来定义遗传密码。这个过程是这个项目的重点。准确的氨基酰基-tRNA合成通常需要许多AARs固有的额外编辑活动。编辑显著减少了在试管中合成氨基酰-tRNA过程中的错误频率，尽管关于反应机制和在活细胞中编辑的影响的许多细节仍不清楚。该项目的目标是阐明编辑的分子机制及其在微生物模式生物(枯草芽孢杆菌、大肠杆菌和酵母)细胞中的生理作用。该项目将开发苯丙氨酰-tRNA合成酶(PHERS)作为一个实验系统，测量翻译中氨基酸选择过程中的错误率，并在体外剖析错误纠正的机制和动力学，为合理设计错误率较高和较低的PHERS变体提供依据。经过改造的PHERS变体将被用来测量活细胞的错误率，并探索它们在不同生长条件下的生物学效应。这项研究的结果将为如何在分子水平上保持遗传密码的准确翻译，以及翻译准确性的变化如何在不同条件下影响细胞的健康和适合性提供新的见解。广泛的影响：该项目将为本科生和研究生提供综合的实验室研究和培训计划，包括历史上代表性不足的少数民族成员。这些努力的影响将通过在地方和国家科学会议上提交研究结果、学生未来的安置情况以及学生对同行评议出版物的作者来衡量。该项目还将为高中教师及其学生提供实验室培训和教育经验。这将包括有指导的小学实验室研究培训，并将在由Westville City School和Metro High School组织的俄亥俄州高中生STEM研究论坛上介绍研究结果。这次会议对公众开放，由在当地大学或公司从事研究的高中生发表演讲。此外，研究生导师和PI都以评委的身份参加这次会议，就研究的成功提供反馈，并有机会与公众讨论现代微生物分子生物学研究。评估的有效性将通过学生、他们的老师和家长的反馈，学生在STEM学位课程中的未来安置，以及学生对同行评议出版物的作者来评估。

Intellectual Merit:To function properly the cell must maintain a certain level of fidelity in all processes dealing with the transfer of genetic information. Translation is the process by which genetic information encoded in a nucleic acid template sequence is read to make a protein having the corresponding amino acid sequence. The fidelity of translation is determined by the accuracy of two key processes: the synthesis of cognate amino acid/tRNA pairs by aminoacyl-tRNA synthetases (aaRS) and the selection of the correct aminoacyl-tRNA by the ribosomes to match the next codon on the mRNA template. The aaRSs define the genetic code by pairing tRNAs with the corresponding amino acids. This process is the focus of this project. Accurate aminoacyl-tRNA synthesis often requires an additional editing activity intrinsic to many aaRSs. Editing significantly decreases the frequency of mistakes during aminoacyl-tRNA synthesis in the test tube, although many details of the reaction mechanism and of the impact of editing in living cells remain unclear. The goals of this project are to clarify the molecular mechanisms of editing and its physiological role in the cells of microbial model organisms (Bacillus subtilis, Escherichia coli and yeast). The project will develop phenylalanyl-tRNA synthetase (PheRS) as an experimental system to measure the error rate during amino acid selection in translation and to dissect the mechanism and kinetics of error correction in vitro, providing a basis to rationally engineer PheRS variants with both higher and lower error rates. The engineered PheRS variants will be used to measure error rates in living cells and probe their biological effects under different growth conditions. The results of this research will provide new insights into how accurate translation of the genetic code is maintained at the molecular level, and how changes in the accuracy of translation impact the health and fitness of cells under different conditions.Broader impacts:The project will provide integrated laboratory research and training programs for undergraduate and graduate students, including members of historically underrepresented minorities. The impact of these efforts will be measured by the presentation of research findings at local and national scientific meetings, by future placements of students, and by student authorship of peer reviewed publications. This project will also provide laboratory training and education experiences for high school teachers and their students. This will involve mentored primary laboratory research training, and will culminate with presentation of the research findings at an Ohio high school student STEM research forum entitled "Bodies", which is organized by Westerville City Schools and Metro High School. This meeting is open to the public and features presentations by high school students who have undertaken research at local universities or companies. In addition, both the graduate student mentor and PI participate at this meeting as judges providing feedback on the success of the research and the opportunity to discuss modern microbial molecular biology research with the general public. The effectiveness of this will be assessed by feedback from the students, their teachers and parents, future placements of students in STEM degree programs, and by student authorship of peer reviewed publications.