Project Summary: Organismal growth and viability is dependent on the faithful and fast decoding of its genomic information into functional peptide sequences. High-accuracy protein synthesis ensures that errant polypeptides, which are more prone to misfold and hence may have undesirable toxic consequences, are not produced. The overall fidelity of protein synthesis appears to be limited by the action of the ribosome, which is the two-subunit macromolecular machine responsible for the decoding of the messenger RNA into protein in all domains of life. During each cycle of elongation, the ribosome carefully selects the appropriate aminoacyl-tRNA (aa-tRNA) that matches the codon in the decoding center from a large-pool of competing aa-tRNAs. In addition to this, we have recently uncovered a quality control mechanism on the ribosome that takes place after peptide-bond formation, which contributes to high-fidelity protein synthesis. Akin to the proofreading strategies enjoyed by DNA and RNA polymerases and tRNA synthetases, the newly discovered ribosome-based mechanism is in place to monitor the quality of the just completed chemical step. During the elongation cycle, the incorporation of an incorrect amino acid was found to have dramatic effects on the specificity of the subsequent reaction. This iterated accumulation of errors results in the abortive termination of protein synthesis by release factors, which under normal conditions rarely decode sense codons. The long term goal of our work 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 to the decoding center, which 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 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 low-resolution structural probing techniques. As a third goal we are interested in exploring a previously unknown role for release factor 3 in the quality control mechanism and its utility in cellular viability. Finally we are interested in finding whether this system exists in eukaryotes, and identifying other factors, if any, that might be involved during this process.

Project Narrative: Recombinant protein technologies are at the forefront of the process by which many therapeutic agents are produced. Information obtained from the research proposed here has immediate ramifications for the means by which this process is currently carried out, especially that the quality and yield of over- expressed proteins appear to be intimately correlated. Furthermore, as the ribosome is the target of many antibiotics, the proposed research is likely to shed light into their mode of action in order to make more effective therapeutics. __SpecificAimsTextDelimiter__ A. Specific Aims The ribosome is arguably one of the most complex machines in biology and is responsible for protein synthesis in all domains of life. Recent progress in structural, biochemical and computational approaches has added much to our understanding of its function, with the discovery that the ribosome is a ribozyme being one of the greatest breakthroughs in this decade. These advances in analytical tools also make it an exciting time to be studying the ribosome. Our lab has made significant contributions towards understanding the mechanisms of translational fidelity. In particular we have identified a quality control mechanism by which the ribosome, like DNA and RNA polymerases, can backtrack to check if mistakes have occurred after peptide-bond formation had taken place. More specifically, we showed that the ribosome monitors the tRNA:mRNA interaction in the P site, and if this interaction is non-canonical, the subsequent decoding step in the A site loses its specificity for cognate substrates and ultimately leads to premature termination of the miscoded polypeptide. The long- term goal of our work is to gain a thorough understanding of the molecular mechanisms underlying this process. A1: To define features of the ribosome and release factors that are important for the post-peptidyl transfer quality control mechanism. As a first step, a kinetic and thermodynamic framework will be established for the normal peptide release reaction, i.e. on stop codons, using a range of pre-steady techniques that include rapid quench and fluorescence stopped-flow experiments. The parameters obtained for the canonical release reaction will in turn be directly compared to those determined for ribosomal complexes containing a perturbed P site. This framework will also be used to evaluate the effects of ribosome mutations on both canonical and non-canonical release reactions. As for tRNA-selection, we have recently undertaken a detailed kinetic study to characterize steps in the tRNA selection pathway that are affected by mismatches in the P site and compared them to those determined for ram and restrictive ribosomes. We plan to conduct similar experiments with ribosome variants that are proposed to alter the interface between the P and A sites. Finally, low-resolution footprinting structural approaches will be used to assess the structure of the A site in the presence of a mismatched P site. The results obtained from these experiments will be used to make mutant ribosomes for RF- and tRNA-selection analysis. These combined studies on RF- and tRNA-selection are likely to shed new light on how signals are communicated between the P site and the decoding center. A2: To determine the role of release factor 3 (RF3) during protein synthesis. Our investigation of the post-peptidyl transfer quality control mechanism revealed that RF3 significantly stimulates the premature termination of protein synthesis on ribosomes containing a mismatched P site. We propose to knock out RF3 in a variety of bacterial strains that have pre-existing defects in protein synthesis accuracy. The phenotype of the resulting strains will be assessed under a range of conditions, especially conditions that may affect translational accuracy, and compared to those exhibited by wild-type E. coli. The quality of proteins in these strains will also be examined using analytical tools such as functional assays, mass spectrometry, and 2D-gel electrophoresis. The yield of protein synthesis will be determined using reporter proteins that contain problematic codons in their coding sequence. A3: To determine whether the post-peptidyl quality control mechanism exists in eukaryotes. The mechanism of translation is highly conserved along the evolutionary tree. We plan to investigate the existence of a ribosomal quality control mechanism in yeast by employing reporter genes programmed to be incorrectly decoded at specific codons during translation and looking for peptide products resulting from premature termination. If the quality control mechanism does exist in yeast, we are interested in identifying factors that might be involved in the process through the use of purified yeast translational machinery.