TETHERED FE(II)--A STRUCTURAL PROBE OF RIBOSOMAL RNA

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The research proposed here involves covalently attaching Fe(II)-EDTA probes to ribosomal RNA (rRNA) in order to determine the tertiary structure of highly conserved regions of the RNA. It is part of a larger project designed to produce the most accurate three dimensional model of E.Coli ribosomes to date. Ribosomes perform protein synthesis, which is one of the most basic aspects of life. Among other functions, protein synthesis has been shown to be part of the cell cycle. Knowing the tertiary structure of ribosomes will help elucidate the active sites of ribosomes and the mechanism of translation, which should aid in finding ways to arrest the cell cycle and provide a treatment for cancer. Two methods of attaching Fe(II)-EDTA to rRNA are proposed: 1) Fe(II)-EDTA will be attached to a 5'-thiophosphate creating a discontinuity in the RNA. 2) Fe(II)-EDTA will be attached to a modified uridine generating a continuous RNA molecule. The cleavage patterns of the modified RNA will be used to determine what parts of the 23S rRNA contact the 16S rRNA at the interface between the 30S and 50S subunits of ribosomes, and to ascertain what regions of 16S rRNA are close to the 530 stem-loop, specifically determining if the 1400-1500 decoding region is proximal to this loop.