STRUCTURE AND FUNCTION OF NUCLEIC ACIDS

真核基因含有内含子， 遗传信息。 内含子从RNA转录本中去除， 通过RNA剪接。 该项目继续调查 酵母中两种RNA剪接的剪接机制：tRNA和mRNA。 tRNA前体在两步反应中剪接。 核酸内切酶 移除内含子，连接酶需要ATP将外显子连接在一起。 将核酸内切酶纯化至均一。 的机制 将研究它识别和切割前体。 连接酶具有 已经被提纯到同质化。 进一步的研究将探讨其 结构域结构和连接酶反应机理。 研究a 合成的tRNA-Phe前体将阐明第二和 底物识别中成熟结构域的三级结构。 mRNA前体在称为剪接体的大颗粒上剪接。 已经分离出称为rna 2 -11的酵母突变体，其定义了 剪接体 RNA基因产物将使用体外纯化方法纯化。 互补分析 这些产品，一旦纯化，可用于 定义和表征的其他RNA和蛋白质组分， 剪接体 剪接体将被纯化并表征为： 电子显微镜和生化分离。 它所通过的途径 是组装和拆卸将被确定。 可能发挥的作用 也将研究mRNA剪接中的hnRNP。

Eukaryotic genes contain introns which interrupt the continuity of the genetic information. Introns are removed from an RNA transcript of the gene by RNA splicing. This project continues an investigation of the mechanism of splicing of two kinds of RNA splicing in yeast: tRNA and mRNA. tRNA precursors are spliced in a two-step reaction. An endonuclease removes the intron and a ligase, requiring ATP, joins the exons together. The endonuclease will be purified to homogeneity. The mechanism by which it recognizes and cleaves the precursor will be studied. The ligase has already been purified to homogeneity. Further studies will explore its domain structure and the mechanism of the ligase reaction. Studies on a synthetic tRNA-Phe precursor will elucidate the role of secondary and tertiary structure of the mature domain in substrate recognition. mRNA precursors are spliced on a large particle called the spliceosome. Yeast mutants have been isolated called rna2-11 which define components of the spliceosome. The RNA gene products will be purified using an in vitro complementation assay. These products, once purified, can be used to define and characterize other RNA and protein components of the spliceosome. The spliceosome will be purified and characterized by electron microscopy and biochemical fractionation. The pathway by which it is assembled and disassembled will be determined. A possible role of hnRNPs in mRNA splicing will also be investigated.