A Second Divalent Metal Ion in the Group II Intron Reaction Center

Abstract

Group II introns are mobile genetic elements that have been implicated as agents of genetic diversity, and serve as important model systems for investigating RNA catalysis and pre-mRNA splicing. In the absence of an atomic-resolution structure of the intron, detailed understanding of its catalytic mechanism has remained elusive. Previous identification of a divalent metal ion stabilizing the leaving group in both splicing steps suggested that the group II intron may employ a "two-metal ion" mechanism, a catalytic strategy used by a number of protein phosphoester transfer enzymes. Using metal rescue experiments, we now reveal the presence of a second metal ion required for nucleophile activation in the exon-ligation step of group II intron splicing. Coupled with biochemical and structural evidence of at least two metal ions at the group I intron reaction center, these results suggest a mechanistic paradigm for describing catalysis by large ribozymes.

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

“…Such a rescue is also part of so-called nucleotide analogue interference mapping/suppression (NAIM/NAIS) experiments, which are used to identify catalytically crucial atoms within a RNA and/or tertiary contacts [253][254][255]. This technique has been widely applied to study metal ion binding sites and their influence on catalytic RNAs, i.e., ribozymes [256][257][258][259][260][261][262]. Major limitations of this method are the fact that (i) T7 RNA polymerase, which is generally used for the transcription of defined RNA sequences, inserts only S p thionucleotides under inversion of their conformation to R p .…”

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pKa

“…Typically, the observed rate of a cleavage reaction is much faster in divalent than in monovalent ions. Hypothetically, this might be due to a direct increase in the rate of the bond-breaking step if, for example, the divalent ion were specifically positioned to neutralize the developing negative charge on the phosphate during the transition state or interact with the 29 or 59 oxygens, as has been found for other ribozymes, such as Group I and II introns (Shan et al 1999;Zhang and Doudna 2002;Stahley and Strobel 2005;Gordon et al 2007). Alternatively, the divalent ion may play an indirect role, such as in positioning of the catalytic functional groups, in a variety of electrostatic effects in the transition state and/or ground state, or in modulating the pK a of a functional group involved in general acid or general base catalysis (Fedor 2002;Emilsson et al 2003;Kraut et al 2003;Lonnberg and Lonnberg 2005;Hoogstraten and Sumita 2007;Scott 2007;Sigel and Pyle 2007).…”

Influence of decreased solvent permittivity on the structure and magnesium(II)-binding properties of the catalytic domain 5 of a group II intron ribozyme

“…There are numerous studies that deal with the metal ion-binding properties of group II introns [7,10,14,[29][30][31][32][33][34][35]. These studies were usually performed in aqueous buffer solutions exhibiting a dielectric constant (permittivity) close to the one of water (ε ≈ 78.5; 25 °C).…”