The long-term goal of this proposal is to identify and understand the complex system of protein translation in terms of its molecular interactions. Eukaryotic translation has been studied since the 1970s using traditional biochemical and genetic approaches. We have recently demonstrated that a new modern approach based on the use of complete genome sequences arid proteomics can shed new light on this complex and essential cellular process. I have pioneered a proteomics approach called """"""""Direct Analysis of Large Protein Complexes"""""""" or DALPC to directly identify novel proteins associated with translational complexes. This highly sensitive mass spectrometry method is capable of comprehensively identifying 100 individual proteins in complex mixtures present at nanogram levels without the need for separating the proteins by gel electrophoresis. This approach has already identified a novel component of the 40S core ribosomal subunit. Starting with yeast Saccharomyces cerevisiae, my strategy is to purify translational protein complexes and to comprehensively identify the proteins using mass spectrometry-based methods. Translation complexes will be isolated using multiple approaches. First, actively translating nbosome subunits and associated translation factors will be separated by sucrose gradient fractionation of cell lysates. Second, translation factors will be stripped off active ribosomes using high salt washes and separated from the core ribosome particles by centrifugation. Finally, affinity-tagged translation proteins will be purified under non-denaturing conditions to isolate physically associated proteins. For all three approaches, the components will be identified using the DALPC approach. Novel proteins identified will be analyzed using a series of bioinformatics tools and data repositories to prioritize the proteins for further characterization. Genetic and biochemical experiments will then be used to validate whether novel proteins found co-purifying with translational complexes are actually involved in translation. Specifically, deletion or conditional mutants will be obtained and compared to isogenic wild type strains for changes in protein synthesis activity, polyribosome profiles, and sensitivity to protein inhibitors. The success of these experiments will greatly increase our knowledge of the all-important process of protein synthesis.
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