具鋅離子相依性之磷酸硫脂水解核酸酶的結構探討

本研究探討本實驗室之前以人為選擇和演化(in vitro selection and evolution)篩選出具鋅二價離子相依性，且具有催化水解磷酸硫酯鍵活性的核酸酶TW17 RNA之結構。為了充分了解TW17核酸酶的結構特性，我們利用了定點突變(site-directed mutagenesis)並進一步以動力學研究分析，並也採用structural probing 分析RNA的二級結構。這些方法讓我們可將TW17核酸酶的二級結構區分出三個stem結構(S1、S2、S3)和四個loop結構(L1、L2、L3、L4)。並且再根據structural probing分析，我們證實TW17核酸酶上辨識受質的位置在一個鬆散不扎實的大型loop結構(L4)當中。以上重要結果提供了我們進一步去研究TW17核酸酶的Michaelis-Menton kinetics的基礎。在Michaelis-Menton kinetics研究中，我們將TW17核酸酶分割成兩部分，分別是具有催化功能的酵素端，和與受質鍵結的18 mer長度的片段。經過in trans 動力學分析的結果發現，此TW17核酸酶的in trans催化系統之催化效能不佳，但此催化活性的表現也間接證實了上述TW17核酸酶二級結構分析的結果之正確性。

關鍵字

水解磷酸硫酯鍵；二級結構； in trans

並列摘要

The purpose of this research is to decipher structural properties essential to the catalysis of a phosphorothioate thiolesterase RNA previously in vitro isolated from a RNA pool. This phosphororthioate thiolesterase RNA is called the TW17 ribozyme. The secondary structure of the TW17 ribozyme was probed by site-directed mutagenesis accompanying kinetic assays and RNA structural probing analysis. The determined secondary structure for the TW17 ribozyme reveals its composition of three separate stem (S1, S2 and S3) structures with loops (L1, L2, L3 and L4) of variable lengths connecting each other. Results from further RNA structural probing analysis indicated that the TW17 ribozyme substrate binding site locates in a long and flexible loop structure of L4 in the TW17 ribozyme. The important structural information for the TW17 ribozyme provides us the blueprint to dissect the ribozyme into an in trans catalysis system capable of performing the classical Michaelis-Menton kinetics. Analysis of the in trans TW17 ribozyme system revealed its poor catalysis performance as evidenced by its low kcat/KM value. The acquired Michaelis-Menton kinetics data, however, validate the accuracy of our resolved secondary structure for the TW17 ribozyme.