Ando Tomoaki, Tanaka Terumichi, Kikuchi Yo
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580.
J Biochem. 2003 Apr;133(4):445-51. doi: 10.1093/jb/mvg058.
The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.
细菌核糖核酸酶P核酶在体外能够将带有CCA-3'标签序列的发夹RNA以及三叶草型前体tRNA作为底物，但具体细节尚不清楚。通过使用反义引导DNA技术改变tRNA结构，我们研究了大肠杆菌核糖核酸酶P核酶对特定形状底物RNA的特异性。对不同镁离子浓度下核糖核酸酶P反应的分析表明，在镁离子浓度低于10 mM时，核酶仅切割三叶草型RNA。在镁离子浓度为10 mM或更高时，核酶也能切割带有CCA-3'标签序列的发夹RNA。在镁离子浓度高于20 mM时，该酶在tRNA衍生发夹中切割位点出现摆动，酶的底物特异性变得更宽泛。使用另一种发夹底物进行的进一步研究也显示了相同的趋势。我们的数据有力地表明，提高金属离子浓度会诱导RNA酶发生构象变化。
