The detailed mechanisms of ribosome function will only be learned by probing the dynamic ribosome at various stages in its translational cycle. We have previously used 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 regions of rRNA near the site of the hybridized DNA oligomer. The approach is to place a cleavage reagent,1,10 orthophenanthroline-Cu(II), at specific sites on DNA oligomers and identify cleavage sites on nearby rRNA, as a result of cleavage induced by this reagent. Phenanthroline-Cu(II) will be bound to various positions on a DNA oligomer, the oligomer hybridized to a targeted, complementary site on the rRNA in the ribosome, and cleavage induced. The resulting cleavages of rRNA will identify the portions of rRNA near the cleavage reagent during various translational stages. The results will not only allow us to identify near-neighbor rRNA/rRNA interactions, but will provide information about the tertiary structure of rRNA itself, and possible conformational changes within this structure. To provide more exact distance mapping, we will place phosphorothioate moieties at specific sites within rRNA, reconstitute the ribosomal subunits, conjugate phenanthroline-Cu(II) to the phosphorothioate and induce cleavage of neighboring rRNA. We will also continue studies on the rRNA regions surrounding tRNA by placing phosphorothioate between targeted nucleotides in tRNA, conjugating phenanthroline-Cu(II) to the thiol group and inducing cleavage of rRNA when the tRNA is in various translational states on the ribosome. By using phenanthroline-Cu(II) having tethers of various lengths, it will be possible to identify neighboring rRNA sites at defined distances from the site where phenanthroline-Cu(II) is attached.
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