Koichi Yoshinari

Mechanistic Studies on Hammerhead Ribozymes

Catalytic RNAs include group I and II introns; the RNA subunit of RNase P; hammerhead, hairpin, and hepatitis delta virus ribozymes; and ribosomal RNA (Altman 1989; Cech 1989; Michel et al. 1989; Noller et al. 1992; Symons 1992; Bratty et al. 1993; Gesteland and Atkins 1993). Of all these catalytic RNAs, the hammerhead ribozyme is the smallest. Naturally occurring hammerhead ribozymes were identified initially within RNA viruses and they act in cis during viral replication by the rolling circle mechanism (Symons 1989, 1992; Bratty et al. 1993). Hammerhead ribozymes have been engineered in such a way that they can act in trans to cleave other RNA molecules (Uhlenbeck 1987; Haseloff and Gerlach 1988). The trans-acting hammerhead ribozyme developed by Haseloff and Gerlach (1988) consists of an antisense section (stems I and III) and a catalytic domain with a flanking stem II and loop section (Fig. 1). Because of the small size of hammerhead ribozymes, they are very suitable for mechanistic studies, being good representatives of catalytic RNA.

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G8 and A9, a site that is considered to play a crucial role in catalysis, was examined by high‐resolution 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the 31P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. 31P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S–substrate complexes generated in the absence of Mg2+ ions were still in an inactive form and that Mg2+ ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

Significant activity of a modified ribozyme with N7-deazaguanine at G10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Several reports have appeared recently of experimental evidence for a double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme‐mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell‐shaped curve for activation of cleavage was used to support the proposed double‐metal‐ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro‐Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (≈20 Å) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro‐Rp phosphoryl P9 oxygen but not with the pro‐Rp phosphoryl oxygen at the cleavage site.

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.

Magnesium-Mediated Cleavage of Phosphorus-Oxygen Bond: A Ribozyme Reaction

Abstract The hammerhead ribozyme belongs to the class of molecules known as antisense RNAs. However, because of short extra sequences that form the so-called catalytic loop, it can act as an enzyme. Since the catalytic domain captures Mg2+ ions and Mg2+ ions can cleave phosphodiester bonds, hammerhead ribozymes are recognized as metalloenzymes. In general, the cleavage of phosphodiester bonds involves acid/base catalysis, with proton transfer occurring in the transition state. When the possibility of such a proton-transfer process was examined by measuring solvent isotope effects, it became apparent that no proton transfer occurs in the transition state during reactions catalyzed by a hammerhead ribozyme. It is likely, therefore, that hammerhead ribozymes exploit the general double-metal-ion mechanism of catalysis, with Mg2+ ions coordinating directly with the attacking and leaving oxygen moieties. Moreover, NMR data suggest that Mg2+ ions are not only important as the true catalysts in the function of ri...