Hammerhead ribozymes: importance of stem-loop II for activity.
The activity of several hammerhead ribozyme constructs with constant lengths of stems I and III of 5 nt each but with variously shortened stems II is reported. Stems with 2 bp rather than the conventional 4 bp show essentially unaltered catalytic activity, independent of the composition of the tetraloop. Further reduction in size to 1 bp or 0 bp decreases activity drastically. Inversion of the G10.1.C11.1 bp next to the invariant core leads to a loss in activity, even when the stem consists of 4 bp. Thus, the minimal structural requirement for stem-loop II is a 2-bp stem with a conserved G.C bp. The reduction in catalytic activity is predominantly a result of a decrease of catalytic constant kcat, whereas Km is only slightly affected. Thus, the structural requirement for optimal activity in these constructs where the chemical-cleavage step is rate limiting is determined by the stabilization of the transition state.
Tuschl
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Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany.
Eckstein
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D001483:Base Sequence; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D013329:Structure-Activity Relationship
D016337:RNA, Catalytic