In vitro selection and characterization of a novel Zn(II)-dependent phosphorothiolate thiolesterase ribozyme

Tzu Pin Wang*, Yu Chih Su, Yi Chen, Yi Ming Liou, Kun Liang Lin, Eng Chi Wang, Long Chih Hwang, Yun-Ming Wang, Yen Hsu Chen

*Corresponding author for this work

Research output: Contribution to journalArticle

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Abstract

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 Mg2+ for maximal catalysis. The TW17 ribozyme, in the presence of Zn 2+ and Mg2+, conferred a rate enhancement of at least 5 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.

Original languageEnglish
Pages (from-to)496-510
Number of pages15
JournalBiochemistry
Volume51
Issue number1
DOIs
StatePublished - 10 Jan 2012

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    Wang, T. P., Su, Y. C., Chen, Y., Liou, Y. M., Lin, K. L., Wang, E. C., Hwang, L. C., Wang, Y-M., & Chen, Y. H. (2012). In vitro selection and characterization of a novel Zn(II)-dependent phosphorothiolate thiolesterase ribozyme. Biochemistry, 51(1), 496-510. https://doi.org/10.1021/bi201585d