An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction

Abstract

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-S_P position of the reactive phosphoryl group is rescued by Mn²⁺. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn²⁺ interaction with the S_P sulfur. Instead, the apparent rescue arises from a Mn²⁺ ion interacting with the residue immediately 3′ of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca²⁺ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn²⁺ at site D are favorable for the chemical reaction, the Ca²⁺ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion–RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.