PROBIG RIBOSOMAL FUNCTION

核糖体功能的详细机制只有通过 在其翻译周期的各个阶段探测动态核糖体。 我们一直在使用短的互补DNA寡聚体来评估 核糖体RNA的各个区域在不同阶段的可用性 翻译.我们现在建议扩展这些研究， 当tRNA结合时，tRNA与rRNA、mRNA和其他tRNA的相互作用 到核糖体。该方法是将切割试剂，或者1，10 邻二氮杂菲-Cu（II）或Fe（II）-EDTA在tRNA上的特定位点， 鉴定附近rRNA、mRNA或相邻tRNA上的切割位点 由这些试剂引起的分裂。菲咯啉，在 Cu++在还原性环境中，导致附近的核酸断裂 acids. EDTA在Fe++存在下，在类似条件下， 相同，但通过不同的机制。这些提供了强大的、特定于站点的 用于探测与复杂位点相邻的RNA区域的工具。我们将约束 切割试剂到tRNA上的各种选定位置，然后 复合的tRNA将以特定的状态与核糖体结合， 卵裂诱导。由此产生的核糖体RNA裂解将识别 切割试剂附近的核糖体RNA部分。我们还将 检测相邻的转运RNA和信使RNA的剪切。的 rRNA的结果不仅可以让我们识别近邻 tRNA/rRNA相互作用，但有助于将tRNA定位在核糖体上， 提供rRNA自身三级结构的信息。的 结果与相邻的tRNA分子将有助于表征其时间 因为它们与tRNA和核糖体结合。结果 将有助于mRNA相对于tRNA的位置 分子。所有这些方法都将使人们更好地了解 核糖体的翻译功能。

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.