In protein synthesis, the information present in messenger RNA (mRNA) sequences is translated into proteins in an essential process carried out by ribosomes in all living organisms. The termination of translation, a crucial step in protein synthesis, must occur with high fidelity, to prevent premature release from the ribosome of incompletely synthesized proteins. Release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the mRNA and trigger hydrolysis of the bond linking the newly completed protein to the tRNA in the P site. The long-term objective of this research is to study the mechanism of translation termination to determine how high fidelity is achieved. Examination of crystal structures of RF1 and RF2 bound to the ribosome has revealed amino acids in the RFs that are crucial for stop codon recognition. The first aim of the project is to probe how these key residues dynamically discriminate between sense and stop codons. In the second aim, the project will examine the roles of conformational changes that RF1 and RF2 undergo when they bind to the ribosome. These conformational changes may serve as functional switches to activate hydrolysis of peptidyl-tRNA linkages after stop codons are recognized. Currently there is no information available about the conformational changes that occur in RF1 and RF2 as they bind to the ribosome. In the third aim, the mechanism by which release factor 3 (RF3) catalyzes the dissociation of RF1 and RF2 from the ribosome will be elucidated. This research will investigate these outstanding questions in translation termination using fluorescence-based, pre-steady state kinetic analysis, site-directed mutagenesis of critical residues, and biochemical assays.
Broader Impacts: This project serves an important educational need by integrating research and teaching. Several undergraduate minority students and graduate students will obtain state-of-the-art research training in advanced biophysical methods. The PI actively participates in outreach activities such as the STARS program and the UCSD Next Step program. In addition, the PI has mentored students from the local high schools to carry out science projects in the laboratory. A high school student from Canyon Crest Academy will join the PI's laboratory this summer. The undergraduates and high school student will carry out site-directed mutagenesis of RF1 and RF2 and will also assist in the purification and characterization of mutant proteins, to contribute to the work while gaining important scientific skills. Several undergraduates have contributed significantly in the past and co-authored research publications. The results of this study will also be published in scientific journals and disseminated broadly to enhance scientific understanding of protein synthesis, a central step in gene expression.
