The detailed mechanisms of ribosome function will only be learned by probing the dynamic ribosome at various stages in its translational cycle. We have been using short, complementary DNA oligomers to assess the availability of various regions of ribosomal RNA at different stages of translation. We now propose to extend these studies by looking at the interaction of tRNA with rRNA, mRNA and other tRNAs as the tRNA is bound to the ribosome. The approach is to place a cleavage reagent, either 1,10 orthophenanthroline-Cu(II) or Fe(II)-EDTA at specific sites on tRNA and identify cleavage sites on nearby rRNA, mRNA or adjacent tRNA as a result of cleavage induced by these reagents. Phenanthroline, in the presence of Cu++ and in a reducing environment, causes scission of nearby nucleic acids. EDTA in the presence of Fe++, under similar conditions, does the same, but via a different mechanism. These provide powerful, site-specific tools to probe RNA regions adjacent to complexed sites. We will bind the cleavage reagents to various, selected positions on tRNA, following which the complexed tRNA will be bound to the ribosome in a specific state, and cleavage induced. The resulting cleavages of ribosomal RNA will identify the portions of ribosomal RNA near the cleavage reagent. We will also assay the neighboring transfer RNA and messenger RNA scissions. The results with rRNA will not only allow us to identify near-neighbor tRNA/rRNA interactions, but will help position tRNA on the ribosome and provide information about the tertiary structure of rRNA itself. The results with adjacent tRNA molecules will help characterize their temporal relations as they are bound to the tRNA and to the ribosome. The results with mRNA will aid in the placement of the mRNA relative to the tRNA molecules. All of these approaches will provide increased understanding of the ribosome in its translational functions.
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