The longest-running current project in the lab is the study of translation termination in a bacterial system. Translation termination takes place when one of three stop codons is recognized in the A site of the ribosome by a class 1 release factor (RF) protein. The protein RFs are bifunctional molecules, like tRNAs. One functional end of the RFs carries a "tripeptide anticodon" motif that is responsible for deciphering stop codons in the small subunit decoding center. The other end carries a glycine-glycine-glutamine "GGQ" motif that assists in catalyzing the hydrolytic reaction that releases the nascent polypeptide chain. We are interested in understanding how RFs carry out this catalysis and also how RFs achieve such remarkably high fidelity greater fidelity than even the process of tRNA selection.
To study these questions we utilize pre-steady state kinetic equipment, including rapid quench and stopped-flow, as well as developing a structural probing approach through site-directed probing with hydroxyl radicals (He and Green, NSMB, 2010), and fluorescence techniques. Work from our lab has clarified the role of the GGQ motif in the catalysis of peptidyl-tRNA hydrolysis, the critical role of the peptidyl-tRNA 2 hydroxyl in orienting the nucleotide (Shaw et al., Chem Biol, 2012; Brunelle et al., RNA, 2008; Youngman et al., Mol Cell, 2007), and the structural mechanism of the remarkably high fidelity RFs (Youngman et al., Cell, 2004). We are currently working to establish the kinetic framework of highly accurate, RF-catalyzed translation termination.
In addition, our lab recently uncovered a quality control mechanism on the ribosome that takes place after peptide-bond formation and contributes to high-fidelity protein synthesis (Zaher and Green, Cell, 2011; Zaher and Green, Nature, 2009). Akin to the proofreading strategies employed by DNA and RNA polymerases and tRNA synthetases, this newly discovered ribosome-based mechanism is in place to monitor the quality of the just-completed chemical bond. We have found that the incorporation of an incorrect amino acid during one elongation cycle has dramatic effects on the specificity of the next elongation cycle (Zaher and Green, RNA, 2010). This iterated accumulation of errors results in the abortive termination of protein synthesis by the release factors, which under normal conditions rarely induce termination upon sense codons.
Our long term goal for this project is to gain a thorough understanding of the molecular mechanisms underlying this process. Our immediate goal is to find out how the signal is communicated from a perturbed mRNA-tRNA interaction in the ribosomal P site to the decoding center, and how that signal ultimately leads to low-fidelity protein synthesis. We are also interested in how the activity of release factors is modulated on sense codons in the presence of a perturbed mRNA-tRNA interaction, and in the structural cues that are responsible for this activity. These goals are built around pre-steady state kinetics approaches in the context of mutated translation components and around low-resolution structural probing techniques. As a third goal, we are interested in exploring a previously unknown role for release factor 3 in this quality control mechanism and in its utility in cellular viability. We are also interested in determining the extent of evolutionary conservation of this quality control mechanism.