Corroboration of Zn(ii)-Mg(ii)-tertiary structure interplays essential for the optimal catalysis of a phosphorothiolate thiolesterase ribozyme.

The TW17 ribozyme, a catalytic RNA selected from a pool of artificial RNA, is specific for the Zn2+-dependent hydrolysis of a phosphorothiolate thiolester bond. Here, we describe the organic synthesis of both guanosine α-thio-monophosphate and the substrates required for selecting and characterizing the TW17 ribozyme, and for deciphering the catalytic mechanism of the ribozyme. By successively substituting the substrate originally conjugated to the RNA pool with structurally modified substrates, we demonstrated that the TW17 ribozyme specifically catalyzes phosphorothiolate thiolester hydrolysis. Metal titration studies of TW17 ribozyme catalysis in the presence of Zn2+ alone, Zn2+ and Mg2+, and Zn2+ and [Co(NH3)6]3+ supported our findings that Zn2+ is absolutely required for ribozyme catalysis, and indicated that optimal ribozyme catalysis involves the presence of outer-sphere and one inner-sphere Mg2+. A survey of the TW17 ribozyme activity at various pHs revealed that the activity of the ribozyme critically depends on the alkaline conditions. Moreover, a GNRA tetraloop-containing ribozyme constructed with active catalysis in trans provided catalysis and multiple substrate turnover efficiencies significantly higher than ribozymes lacking a GNRA tetraloop. This research supports the essential roles of Zn2+, Mg2+, and a GNRA tetraloop in modulating the TW17 ribozyme structure for optimal ribozyme catalysis, leading also to the formulation of a proposed reaction mechanism for TW17 ribozyme catalysis.

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

Here we present the in vitro selection of a novel ribozyme specific for Zn2+-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 Zn2+ and Mg2+ for maximal catalysis. The TW17 ribozyme, in the presence of Zn2+ 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 Zn2+, like Zn2+-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn2+ in ribozyme catalysts.