Magnesium is essential for formation of an active complex of a hammerhead ribozyme with its substrate: an investigation by NMR spectroscopy.
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 three-base-paired duplex and a four-base-paired duplex that included a wobble 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. 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.
Orita
M
M
Yamanouchi Pharmaceutical Co. Ltd, Tsukuba Science City, Japan.
Vinayak
R
R
Andrus
A
A
Takagi
Y
Y
Chiba
A
A
Kaniwa
H
H
Nishikawa
F
F
Nishikawa
S
S
Taira
K
K
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012313:RNA; D008274:Magnesium