Tuschl T, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany.
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6991-4. doi: 10.1073/pnas.90.15.6991.
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.
报道了几种锤头状核酶构建体的活性，其茎I和茎III的长度恒定，均为5个核苷酸，但茎II的长度不同程度缩短。具有2个碱基对而非传统4个碱基对的茎显示出基本未改变的催化活性，与四环的组成无关。进一步减小尺寸至1个碱基对或0个碱基对会大幅降低活性。紧邻不变核心的G10.1.C11.1碱基对发生倒置会导致活性丧失，即使茎由4个碱基对组成。因此，茎环II的最小结构要求是具有保守G.C碱基对的2个碱基对的茎。催化活性的降低主要是催化常数kcat降低的结果，而Km仅受到轻微影响。因此，在这些化学切割步骤为限速步骤的构建体中，最佳活性的结构要求由过渡态的稳定性决定。
