Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Abstract

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two transacting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg2+, Ca2+ and Mn2+, lower with Co2+ and Sr2+ and some variants are also active with Cd2+ and Zn2+ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg2+ is partially inhibited in the presence of Na+, spermidine and some other divalent metal ions. The inert Co(NH3)63+ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.