Thomson J B, Lilley D M
Department of Biochemistry, The University of Dundee, United Kingdom.
RNA. 1999 Feb;5(2):180-7. doi: 10.1017/s1355838299981670.
In the natural form of the hairpin ribozyme the two loop-carrying duplexes that comprise the majority of essential bases for activity form two adjacent helical arms of a four-way RNA junction. In the present work we have manipulated the sequence around the junction in a way known to perturb the global folding properties. We find that replacement of the junction by a different sequence that has the same conformational properties as the natural sequence gives closely similar reaction rate and Arrhenius activation energy for the substrate cleavage reaction. By comparison, rotation of the natural sequence in order to alter the three-dimensional folding of the ribozyme leads to a tenfold reduction in the kinetics of cleavage. Replacement with the U1 four-way junction that is resistant to rotation into the antiparallel structure required to allow interaction between the loops also gives a tenfold reduction in cleavage rate. The results indicate that the conformation of the junction has a major influence on the catalytic activity of the ribozyme. The results are all consistent with a role for the junction in the provision of a framework by which the loops are presented for interaction in order to create the active form of the ribozyme.
在发夹状核酶的天然形式中，包含大部分活性必需碱基的两个带有环的双链体形成了一个四向RNA连接的两个相邻螺旋臂。在本研究中，我们以一种已知会扰乱整体折叠特性的方式操纵了连接周围的序列。我们发现，用具有与天然序列相同构象特性的不同序列替换连接，会使底物切割反应的反应速率和阿伦尼乌斯活化能非常相似。相比之下，旋转天然序列以改变核酶的三维折叠会导致切割动力学降低十倍。用对旋转成允许环之间相互作用所需的反平行结构具有抗性的U1四向连接替换也会使切割速率降低十倍。结果表明，连接的构象对核酶的催化活性有重大影响。这些结果都与连接在提供一个框架中的作用一致，通过这个框架，环被呈现出来进行相互作用，以形成核酶的活性形式。
