Researches on ribozymes at molecular and atomic level

对天然核酶的分子设计、结构和反应机理进行了研究。确定了I组内含子核酶的最小催化单位。确定了该内含子的RNA折叠途径和分子间长程相互作用。体外筛选技术被成功地用于生产人工核酶和RNPs。锤头状核酶被成功地设计用于体内使用。开发了新的基因发现系统。对哺乳动物线粒体核糖体RNA及其相应的蛋白质组分进行了鉴定和分析。已经阐明，茎蛋白与核糖体GTP酶中心rRNA的结合参与了两个RNA结构域的功能结构的形成。这些复合体不仅影响rRNA中的延伸因子结合部位，而且还影响解码部位的功能结构。克隆了与裂解酵母Prp1相互作用的Prp12基因，Prp1是U4/U6SnRNP的组成部分。结果表明，在剪接的早期阶段，U4/U6 SnRNP与U2 SnRNP的相互作用不仅取决于U2和U6 SnRNAs之间的氢键作用，而且还取决于与SnRNAs结合的蛋白质之间的相互作用。

The molecular design, structure and reaction mechanism of the naturally occurring ribozymes were investigated. The minimal catalytic unit of Group I intron ribozymes was identified. The RNA folding pathways and the intermolecular long-range interactions of the intron were identified. In vitro selection te4chnique was successfully employed for producing artificial ribozymes and RNPs. Hammerhead ribozyme was engineered successfully for in vivo use. New gene discovery system was developed. Ribosomal RNAs of mammalian mitochondria and the corresponding protein components were identified and analyzed. It was elucidated that the binding of stalk proteins to rRNA in the ribosomal GTPase center is involved in formation of the functional structure of two RNA domains. These complexes affected not only elongation factor-binding site in the rRNA but also the functional structure of the decoding site. A gene coding for the Prp12 which interacts with Prp1 (pre-mRNA processing 1) of fission yeast which is a component of U4/U6 snRNP was cloned. As a result, it was elucidated that the interaction of U4/U6 snRNP with U2 snRNP which is necessary for conversion of a commitment complex to a spliceosome at the early stage of splicing depends on not only interaction due to hydrogen bonding between U2 and U6 snRNAs, but also interaction among proteins bound to the snRNAs.