PROBING RIBOSOMAL FUNCTION

描述（改编自申请人的摘要）： 核糖体功能只能通过探测动态核糖体来了解， 翻译周期的各个阶段。我们以前使用过短， 互补DNA寡聚体，以评估不同区域的可用性， 处于不同翻译阶段的核糖体RNA。我们现在建议延长 这些研究通过观察杂交位点附近的rRNA区域， DNA寡聚体。方法是将裂解试剂，1，10 邻菲咯啉-Cu（II），在DNA寡聚体上的特定位点，并鉴定 作为该试剂诱导切割的结果，邻近rRNA上的切割位点。 菲咯啉-Cu（II）将结合到DNA寡聚体上的各个位置， 寡聚体杂交到靶向的，互补位点上的rRNA在 核糖体，并诱导裂解。由此产生的rRNA裂解将鉴定 在各种翻译过程中，靠近切割试剂的rRNA部分 阶段结果不仅使我们能够识别近邻rRNA/rRNA 相互作用，但将提供有关rRNA的三级结构的信息 它本身，以及这种结构中可能的构象变化。提供 更精确的距离映射，我们将硫代磷酸酯部分放置在 rRNA内的特定位点，重组核糖体亚基， 菲咯啉-Cu（II）的硫代磷酸酯，并诱导裂解 邻近的rRNA我们还将继续研究周围的rRNA区域， 通过在tRNA中的靶核苷酸之间放置硫代磷酸酯， 将菲咯啉-Cu（II）缀合至硫醇基团并诱导 当tRNA在核糖体上处于不同的翻译状态时，通过使用 菲咯啉-Cu（II）具有各种长度的系链，将有可能 确定与所述位点相距一定距离的相邻rRNA位点， 菲咯啉-Cu（II）连接。

DESCRIPTION (Adapted from the applicant's abstract): 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.