The overall objective of the proposed research is to elucidate the mechanism of protein biosynthesis in subcellular organelles such as chloroplasts and mitochondria. Our immediate goal is to provide a detailed understanding of the mechanism of chloroplast protein biosynthesis in Euglena gracilis and to compare this system to the translational systems of prokaryotes, eukaryotes and of other organelles. Our first objective is to carry out a detailed analysis of the properties of the chloroplast elongation factors EF-Tu and EF-Ts which have recently been purified in our laboratory. We will investigate the binding of aminoacyl-tRNA to EF-Tu and the allosteric interactions between ligand binding sites on this factor. We will then examine the interaction of chloroplast EF-Tu and EF-Ts and will seek to establish the overall mechanism by which the EF-Tu is recycled during the elongation phases of protein synthesis in this organellar system. Our second objective is to complete the purification of the chloroplast factor that binds the initiator tRNA to the small ribosomal subunit during initiation complex formation and to examine the mechanistic details of this process. In addition, we will complete the purification of the chloroplast ribosome dissociation factor, determine its subunit site of action and examine its role in the initiation process. Finally, we will purify the chloroplast mRNA for the large subunit of ribulose 1,5-bisphosphate carboxylase and investigate the factors required for the binding of natural mRNA to chloroplast ribosomes. These factors will be purified and their roles in the initiation process examined in detail. Our long term goal is to develop a defined in vitro system in which to study the regulation of organelle protein biosynthesis, its coordination with cytoplasmic protein synthesis and its integration into the complex metabolism of the cell.
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