The molecular mechanism of post-peptidyl quality control on the ribosome

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DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.