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Published August 1, 1999 | public
Journal Article

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


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.

Additional Information

© 1999 American Chemical Society. Received February 17, 1999; Revised Manuscript Received May 14, 1999. This work was supported by NIH Grant GM49243 to D.H. We are grateful to J. Piccirilli for helpful discussions, F. Eckstein for the gift of 2'-aminoguanosine, L. Beigelman for oligonucleotide substrates, and members of the Herschlag lab for comments on the manuscript.

Additional details

August 19, 2023
October 18, 2023