Translational Fidelity in Eukaryotes

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ABSTRACT With the availability of atomic scale structures of ribosomes, the next critical task is to link ribosome structure with biological function. At the biological level, we have been exploring how ribosomes recognize termination codons and maintain translational reading frame using translational recoding signals of viral origin. Viral recoding is important for virus propagation, and their mRNA-based recoding signals have proved to be of great utility in elucidating the molecular mechanisms underlying these essential tasks. These studies have been based on the yeast Saccharomyces cerevisiae model eukaryotic organism because it provides researchers with the most advanced, diverse, and robust toolbox available. Further, we have built upon this foundation to develop a robust and synergistic combination of molecular genetic, biochemical, structural, and molecular modeling tools. This has enabled us to show that both the biophysical interactions between ribosomal proteins, rRNAs and tRNAs, and the biochemical properties of ribosome-associated enzymatic activities are important for proper reading frame maintenance and stop codon recognition. On a broader scale, our work is defining the allosteric communication pathways that connect and coordinate different functional centers of the ribosome with one another. The broad goal of this proposal is to further define how ribosome structure influences function. Aim 1 will determine the effects of targeted mutations on yeast ribosome structure and function. Specifically, reverse genetics approaches will be applied to define the functions of specific ribosomal proteins and ribosomal RNAs. These studies include expansion to examine the effects of two ribosome-associated mutations in mammalian systems. The second aim will characterize the interactions between ribosomes the Cricket Paralysis Virus Internal Ribosome Entry Signal (CRPV IRES), and the HIV-1 programmed -1 ribosomal frameshift signal. The proposed collaborations represent logical expansions of our work into new and exciting areas. We also anticipate that during the course of the proposed studies, breakthroughs will continue to be made in the area of ribosome structure, and that new discoveries relevant to ribosomes and disease will be unveiled. The proposed program will position us to quickly capitalize on these, providing a strong foundation for new and unanticipated discovery opportunities. In the end, this work will make significant contributions to the scientific and clinical communities by both deepening our understanding of the relationship between ribosome structure and function, while broadening our view of translational fidelity and disease. PROJECT NARRATIVE Proliferating cells, be they embryonic cells busily creating new persons, T-cells fighting off infection, or cancer cells overwhelming the patient, absolutely require large numbers of highly accurate ribosomes to meet their needs for synthesis of new proteins. Ribosomes, the central component of this process, are complex biological nanomachines composed of many protein and RNA molecules, and the overall goal of the proposed research is to begin to understand how the atomic scale structure of the ribosome ultimately determines its function. A deeper understanding of the relationship between ribosome structure and function will aid the rational design of new classes of drugs designed to target a diverse array of clinical applications including antiviral and antibacterial agents, as well as drugs targeting a diverse array of cancers, developmental disorders, and other critical diseases afflicting society.