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

The presence of catalytic metal ions in RNA active sites has often been inf erred from metal-ion rescue of modified substrates and sometimes from inhib itory effects of alternative metal ions. Herein we report that, in the Tetr ahymena group I ribozyme reaction, the deleterious effect of a thio substit ution at the pro-S-p position of the reactive phosphoryl group is rescued b y Mn2+. However, analysis of the reaction of this thio substrate and of sub strates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the appar ent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions b etween the oligonucleotide substrate (S) and the active site. This metal si te is referred to as site D herein. We also present evidence that a previou sly observed Ca2+ ion that inhibits the chemical step binds to metal site D . These and other observations suggest that, whereas the interactions of Mn 2+ at site D are favorable for the chemical reaction, the Ca2+ at site D ex erts its inhibitory effect by disrupting the alignment of the substrates wi thin the active site. These results emphasize the vigilance necessary in th e 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 int eractions to be revealed and the properties of individual metal-ion sites t o be probed, even within the sea of metal ions bound to RNA.