Catalysis in living cells is carried out by both proteins and RNA. Protein enzymes have been known for over 200 years, but RNA enzymes, or "ribozymes", were discovered only 30 years ago. Developing insight into RNA enzyme mechanisms is invaluable for better understanding both extant biological catalysis as well as the primitive catalysis envisioned in an early RNA-catalyzed life. Natural ribozymes include large RNAs such as the group I and II introns; small RNAs such as the hepatitis delta virus and the hairpin, hammerhead, VS, and glmS ribozymes; and the RNA portion of the ribosome and spliceosome. RNA enzymes use many of the same catalytic strategies as protein enzymes, but do so with much simpler side chains. Among these strategies are metal ion, general acid-base, and electrostatic catalysis. In this Account, we examine evidence for participation of charged nucleobases in RNA catalysis. Our overall approach is to integrate direct measurements on catalytic RNAs with thermodynamic studies on oligonucleotide model systems.The charged amino acids make critical contributions to the mechanisms of nearly all protein enzymes. Ionized nucleobases should be critical for RNA catalysis as well. Indeed, charged nucleobases have been implicated in RNA catalysis as general acid-bases and oxyanion holes. We provide an overview of ribozyme studies involving nucleobase catalysis and the complications involved in developing these mechanisms. We also consider driving forces for perturbation of the pK a values of the bases. Mechanisms for pK a values shifting toward neutrality involve electrostatic stabilization and the addition of hydrogen bonding. Both mechanisms couple protonation with RNA folding, which we treat with a thermodynamic formalism and conceptual models. Furthermore, ribozyme reaction mechanisms can be multichannel, which demonstrates the versatility of ribozymes but makes analysis of experimental data challenging.We examine advances in measuring and analyzing perturbed pK a values in RNA. Raman crystallography and fluorescence spectroscopy have been especially important for pK a measurement. These methods reveal pK a values for the nucleobases A or C equal to or greater than neutrality, conferring potential histidine- and lysine/arginine-like behavior on them. Structural support for ionization of the nucleobases also exists: an analysis of RNA structures in the databases conducted herein suggests that charging of the bases is neither especially uncommon nor difficult to achieve under cellular conditions. Our major conclusions are that cationic and anionic charge states of the nucleobases occur in RNA enzymes and that these states make important catalytic contributions to ribozyme activity. We conclude by considering outstanding questions and possible experimental and theoretical approaches for further advances.
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