부산대학교 도서관

Background: 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 La[sup 3+] ions in the presence of a fixed concentration of Mg[sup 2+] 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 A[sub 9] and to the N7 of nucleotide G[sub 10.1] is critical for efficient catalysis, despite the large distance (approx. 20 Å) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd[sup 2+] ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. Results: In earlier discussions, it was difficult to completely exclude the possibility that La[sup 3+] ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G[sub 10.1]. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd[sup 2+] ions, the added La[sup 3+] ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G[sub 10.1] reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg[sup 2+] ion to N7 at G[s... [ABSTRACT FROM AUTHOR]