Browsing by Author "Liou, Yi-Ming"

Now showing 1 - 1 of 1

In Vitro Selection and Characterization of a Novel Zn(II)-Dependent Phosphorothiolate Thiolesterase Ribozyme
(2012-01-10)
Wang, Tzu-Pin
;
Su, Yu-Chih
;
Chen, Yi
;
Liou, Yi-Ming
;
Lin, Kun-Liang
Here we present the in vitro selection of a novel ribozyme specific for Zn(2+)-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn(2+) and Mg(2+) for maximal catalysis. The TW17 ribozyme, in the presence of Zn(2+) and Mg(2+), conferred a rate enhancement of at least S orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn(2+), like Zn(2+)-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn(2+) in ribozyme catalysts.