Abstract
The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. Elevated concentrations (1.0 M) of monovalent Na+ ions could not induce the formation of the R32-RdC11 complex. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.
Highlights
Self-cleaving hammerhead RNA domains are found in many virus-like plant pathogens, and they catalyze the sequencespecific cleavage of RNA [1,2,3]
In terms of its general structure, the hammerhead ribozyme consists of three base pair stems and a central conserved nucleotide core of two nonhelical segments (Fig. 1)
Because the imino protons can exchange with the solvent water, resonance of an imino proton is observed in the NMR spectrum only if the exchange is slow on the NMR time scale (Ͼmsec)
Summary
Self-cleaving hammerhead RNA domains are found in many virus-like plant pathogens, and they catalyze the sequencespecific cleavage of RNA [1,2,3]. Many NMR studies have been performed in successful attempts to reveal the presence of these three base pair stems (4 –7), but no structural information about nonhelical regions has been obtained. Two crystallographic studies have provided a three-dimensional structure of the hammerhead ribozyme [8, 9]. The role of Mg2ϩ ions in the establishment of an active form of hammerhead ribozymes remains obscure, though recent electrophoretic studies demonstrated that the global conformation of the hammerhead ribozyme folds in response to the concentrations and types of ions present [18]. In order to examine the conformational properties of a 32mer ribozyme (R32) and the further role of Mg2ϩ ions, we analyzed the structure by high resolution NMR spectroscopy. We report here that Mg2ϩ ions can induce the structural change in R32 that is necessary for the interaction between the ribozyme and its substrate (RdC11)
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