POLYSOMES & SUBUNITES STRUCTURE-FUNCTION RELATIONSHIPS

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The structure and function of the bacterial ribosome will be investigated using mutants in 16S and 23S rRNA. The rrnB cistron in a recombinant plasmid will be mutagenized at selected sites using synthetic deoxy-oligonucleotides and the M13 cloning technology. Areas to be investigated include ribosomal protein and mRNA binding sites, regions of subunit interaction, the peptidyl transferase site and binding sites for certain antibiotics. Mutant gene products will be characterized in vivo in a modified maxicell system and in vitro after isolation by thiouridine-mercurated agarose affinity chromatography, gel electrophoresis and sucrose gradient centrifugation. This genetic approach will provide specific information about particular regions of rRNA and increase our understanding of the significant role of rRNA in protein synthesis. The structural arrangement of ribosomal protein S1 on 30S and 70S ribosomes will be studied using monoclonal antibodies to S1 and gel immunoelectrophoresis. This is the first application of an approach to be used to characterize the fine structure of many ribosomal proteins. Structure-function studies of yeast ribosomes will focus on characterization of r-protein-rRNA interactions. Primary binding proteins will be identified and specific attachment sites on rRNA will be determined. This will provide basic structural information for subsequent function studies. The role of eIF2 in the regulation of eucaryotic protein synthesis will be studied in yeast. The Alpha subunit is phosphorylated by the recently isolated enzyme eIF2-kinase. The enzyme will be characterized and factors regulating its activation determined. An additional regulatory role of eIF2-kinase, the phosphorylation of r-proteins, will be explored with particular focus on the effect of phosphorylation on YL4, the 5S RNA binding protein. These experiments will increase our understanding of the role of covalent modification in regulation of translation in eucaryotic cells.