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 is transferred from a nucleic acid sequence into the amino acid sequence of a protein. The fidelity of translation is determined at two major points: the accuracy of aminoacyl-tRNA selection by the ribosomes and synthesis of cognate amino acid/tRNA pairs by aminoacyl-tRNA synthetases (aaRS) in the course of the aminoacylation reaction. The aaRSs define the genetic code by pairing tRNAs with the corresponding amino acids. 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 vitro, although many details of the reaction mechanism and the impact of editing on the living cell remain unclear. The goals of this project are to use phenylalanyl-tRNA synthetase (PheRS) as a model system to clarify the molecular mechanisms of editing, and to investigate the physiological role of editing in the cell. The broader impacts resulting from the proposed activity will be to provide integrated laboratory research and training programs for both undergraduate and graduate students including members of historically underrepresented minorities, and to provide laboratory training and education experiences for middle and high school teachers and their students.
All cells need to ensure the accurate production of new proteins, and cells have several mechanisms to ensure this accuracy. One mechanism involves the aminoacyl-tRNA synthetases (aaRS). The aaRS attach amino acids to their appropriate tRNAs. The aminoacyl-tRNAs can then go to the ribosome and participate in protein translation. The aaRS face a particular problem for accuracy as the amino acids can differ by as little as one hyoxyl group, but putting the wrong amino acid on a tRNA and introducing a single inappropriate hydroxyl group can have devastating consequences for a protein. Thus it is maybe no surprise that aaRS have a second function, beyond charging the tRNA, to edit mischarged tRNsA and hydrolyze the aminoacyl-tRNA bond before these problem tRNAs can reach the ribosome. These editing functions of aaRS are universally conserved throughout life, but studies done to date have not found a clear cellular function for editing by aaRS, as the editing function can be removed from a cell and it grows like its wild-type counterpart in the lab. We have been working to develop methods to probe the function of editing by the aaRS. We are using the bacterium Bacillus subtilis as our model system, as it exhibits many developmental processes when it is placed under starvation, a condition typically encountered in nature. This will allow us to test the hypothesis that editing by aaRS is critical for cells under starvation conditions. We are developing mutant strains of B. subtilis that have a single point mutations in the chromosome that knock-out editing by an aaRS as well as peptide reporters that will allow us to accurately asses the degree of mistranslation that occurs. Through these studies we have laid the framework to answer a longstanding question of what is the importance of editing by aaRS for cellular function, while at the same time we have trained undergraduate and graduate students in the critical thinking process of science.
