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.
Scientific outcomes: The explosion of genomic sequencing has provided scientists with "blueprints for life" for organisms ranging from bacteria to humans. At the same time, we are striving to better understand how the cell goes about ensuring these blueprints are accurately followed during gene expression and protein synthesis. Our particular focus is on one event – the faithful matching of nucleic acids with amino acids at the start of protein synthesis. This is a critical moment in gene expression, as it is the first time at which the genetic code comes into play. Any mistakes at this point, and the final product will not faithfully reflect the blueprints. This project was part of our continuing efforts directed towards understanding the mechanisms that determine how cells ensure the accurate translation of information from genes to proteins, the building blocks of all cells. In particular we have focused on how cells maintain such a high degree of accuracy during this process of translation, and what happens when that goes wrong. Studying how cells maintain accuracy is easiest to do using microorganisms, and for this work we chose two well-known model systems, the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. What we have been able to show is how cells add extra steps to enhance quality control and reduce errors. We also found that different cells can tolerate different mistakes, sometimes to a surprisingly high level, depending on where and how they are growing. This goes against the widely held notion that accuracy and error rates are conserved features of all cells and has broad implications across a range of fields from microbial ecology to pharmaceutical production. Broader impacts: During the course of the project we provided training opportunities for undergraduates under the direct supervision of graduate students, for whom this provided the first experience of mentoring. We also provided training for two masters students, one of whom has graduated and the other of whom is working towards transitioning into s STEM graduate program. We also partnered with the Metro Early College High School to bring one high school student to the lab to perform research in Spring 2010 as part of their Bodies program. The student spent two months working in the lab during which time he cloned, expressed and purified M. mobile PheRS under the daily supervision of a graduate student. The progress of the project was monitored weekly both by Ibba and the director of the Bodies program at Metro. At the end of the lab research portion of the program the student wrote a report and gave a short oral presentation at a local STEM education day.
