PROBING RIBOSOMAL FUNCTION

描述(改编自申请人的摘要)： 只有通过探测动态核糖体才能了解核糖体的功能 在其翻译周期的不同阶段。我们以前使用过Short， 互补DNA寡聚体，以评估不同区域的可用性 核糖体RNA在翻译的不同阶段。我们现在建议延长 这些研究是通过观察杂交体附近的rRNA区域进行的 DNA寡聚体。方法是将裂解试剂1，10 邻菲罗啉-铜(II)，在DNA寡聚体上的特定位置并鉴定 在附近rRNA上的裂解位点，这是该试剂引起的裂解的结果。 邻菲罗啉-铜(II)将结合到DNA低聚体上的不同位置 低聚物杂交到rRNA上的靶向互补位置 核糖体和诱导的卵裂。由此产生的rRNA裂解将识别 在各种翻译过程中靠近裂解剂的rRNA部分 各阶段。结果将不仅使我们能够识别近邻rRNA/rRNA 相互作用，但将提供有关rRNA三级结构的信息 以及该结构内可能的构象变化。要提供 更精确的距离图，我们将把硫代磷酸部分放在 RRNA内的特定位置，重组核糖体亚基，共轭 邻菲咯啉-铜(II)对硫代磷酸根的作用及诱导其裂解 邻近的rRNA。我们还将继续研究周围的rRNA区域。 通过将硫代磷酸放置在tRNA中的靶核苷酸之间， 邻菲咯啉-铜(II)与硫醇基的偶联及诱导裂解 当tRNA在核糖体上处于不同的翻译状态时。通过使用 邻菲咯啉-铜(II)具有不同长度的系链，将有可能 确定与指定距离的站点之间的相邻rRNA站点 邻菲咯啉-铜(II)被固定。

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.