Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3-oxygen of the oligonucleotide substrate and the 3-and 2-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA. D ivalent metal ions play a critical role in catalysis by many RNA and protein enzymes (e.g., see refs. 1-10). Determining the number of metal ions in an enzymatic active site and delineating their catalytic roles are crucial for elucidating the catalytic mechanisms of these enzymes (e.g., refs. 1-3, 5, 9, and 11-34). This presents a formidable challenge, especially for RNA enzymes, as the metal ions that directly participate in the chemical transformation are bound within a sea of metal ions that coat the charged RNA backbone and facilitate RNA folding ( Fig. 1; e.g., see refs. 32-39).The Tetrahymena group I ribozyme (E) derived from a selfsplicing group I intron catalyzes a reaction that mimics the first step of splicing, in which an exogenous guanosine nucleophile (G) cleaves a specific phosphodiester bond of an oligonucleotide substrate (S; Eq. 1; refs. 40-42).Three metal ion interactions contribute to catalysis by this ribozyme (Fig. 2). These interactions were previously identified by modification of specific substrate atoms that alter metal ion specificity; reactions with sulfur-or nitrogen-substituted substrates were severely compromised in Mg 2ϩ but were stimulated by addition of softer metal ions such as Mn 2ϩ (3,5,7,9). A fundamental question that remains unanswered, however, is whether these interactions are mediated by the same or by distinct metal ions.We report here the development of an approach that combines atomic-level substrate modifications with quantitative analyses to determine the affinity of individual metal ions for an enzymatic active site. These affinities provide a fingerprint for each metal ion, allowing distinct metal ions to be distinguished. Using this approach, we have provided evidence for three distinct metal ions within the active site of the Tetrahymena ribozyme. The results and the approach described herein will allow us to further probe the functional consequences of specific metal ion interactions and the catalytic role of individual metal ions, even within the sea of metal ions bound to RNA. Materials and MethodsMaterials. Ribozyme was prepared by in vitro transcription with T7 RNA polymera...

These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.

The Tetrahymena ribozyme is a metalloenzyme that catalyzes cleavage of oligonucleotide substrates by phosphoryl transfer. Thiophilic metal ions such as Mn2+, Zn2+ or Cd2+ rescue the >10(3)-fold inhibitory effect of sulfur substitution of the 3'-oxygen leaving group but do not effectively rescue the effect of sulfur substitution of the nonbridging pro-Sp phosphoryl oxygen. We now show that the latter effect can be fully rescued by Zn2+ or Cd2+ using a phosphorodithioate substrate, in which both the 3'-oxygen and the pro-Sp oxygen are simultaneously substituted with sulfur. These results provide the first functional evidence that metallophosphotransferases can mediate catalysis via metal ion coordination to both the leaving group and a nonbridging oxygen of the scissile phosphate.