Dependence on Mg2+ ions of the activities of dimeric hammerhead minizymes.
A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II. In a previous study we demonstrated that a minizyme with high-level activity forms a dimeric structure with a common stem II [Amontov and Taira (1996) J. Am. Chem. Soc. 118, 1624-1628]. We now demonstrate that the stability of the dimeric structure is influenced by Mg2+ ions. We found that the dependence on Mg2+ ions of the activity of homodimeric minizyme (a dimer with two identical binding sites) has composite biphasic characteristics. When the concentration of Mg2+ ions reached a specific critical level, the dependence on the concentration of Mg2+ ions lost its tendency to reach a plateau. In the case of the heterodimeric minizyme (a dimer with two different binding sites), we investigated the kinetic behavior of two different forms of the dimer, namely, free dimer and the complex of the dimer with an uncleavable substrate. The kinetic behavior of the free heterodimer was very similar to that of the homodimeric minizyme. In contrast, in the presence of the uncleavable substrate at a concentration as high as that of the minizyme, the curve for the dependence on Mg2+ ions showed normal saturation kinetics. While, at low concentrations of Mg2+ ions, the activity of the heterodimers was much higher when the dimeric structure was stabilized by the presence of the uncleavable substrate, at high concentrations of Mg2+ ions, this difference in activity became less and less significant. Thus, high concentrations of Mg2+ ions were able to stabilize the dimeric minizymes in the absence of the uncleavable substrate.
Amontov
S
S
National Institute of Bioscience and Human Technology, Tsukuba Science City, Japan.
Nishikawa
S
S
Taira
K
K
D001483:Base Sequence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D013379:Substrate Specificity
D016337:RNA, Catalytic; D008274:Magnesium