Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Abstract

We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2‘-aminoguanosine (G_ NH+_3), in which the 2‘-OH of the guanosine nucleophile (G) is replaced by an –NH+_3 group. At low concentrations of divalent metal ion (2 mM Mg^(2+)), G_NH+_3 binds at least 200-fold stronger than G or G_(NH2), with a dissociation constant of ≤1 μM from the ribozyme·oligonucleotide substrate·G_NH+_3 complex (E•S•G_(NH+_3). This strong binding suggests that the -NH+_3 group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of G_(NH+_3) to E·S more than 10^2-fold. The Mn^(2+) concentration dependence suggests that MC, the metal ion that interacts with the 2‘-moiety of G in the normal reaction, is responsible for this effect. M_C and G_(NH+_3) compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged -NH+_3 and M_C and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M_C and G_(NH+_3), consistent with a network of interactions involving MC that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound G_(NH+_3) is at least 10^4-fold slower than with G or G_(NH2). These results provide additional support for an integral role of M_C in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the -NH+_3 moiety as an electrostatic probe within a structured RNA.