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。确定了内含子的RNA折叠途径和分子间长程相互作用。利用体外筛选技术成功地生产了人工核酶和核糖核酸酶。锤头状核酶被成功地设计用于体内应用。开发了新的基因发现系统。对哺乳动物线粒体的核糖体RNA和相应的蛋白质组分进行了鉴定和分析。阐明了茎蛋白与核糖体GTP酶中心的rRNA结合参与了两个RNA结构域功能结构的形成。这些复合物不仅影响rRNA中延伸因子的结合位点，而且还影响解码位点的功能结构。克隆了编码Prp 12的基因，该基因与作为U4/U6 snRNP组分的裂殖酵母的Prp 1（前mRNA加工1）相互作用。其结果是，阐明了在剪接的早期阶段，定型复合物向剪接体转化所必需的U4/U6 snRNP与U2 snRNP的相互作用不仅依赖于U2和U6 snRNA之间的氢键作用，还依赖于与snRNA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.