COLLABORATIVE RESEARCH: The Role of Quality Control in Microbial Translation

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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.