Vaish N K, Heaton P A, Eckstein F
Max-Planck-Institut für experimentelle Medizin, Göttingen, Germany.
Biochemistry. 1997 May 27;36(21):6495-501. doi: 10.1021/bi963134r.
The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A9U mutation was observed while in the other two mutations at A9U and U7A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 microM(-1) min(-1), respectively, as compared to 163 microM(-1) min(-1) for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure.
锤头状核酶在核心区域有一个不变的核苷酸序列。为了寻找能够支持三联体GUC之后切割的替代序列，将10个核苷酸的核心区域随机化，并通过转录、逆转录和PCR的重复循环进行体外筛选。每次转录后通过变性聚丙烯酰胺凝胶电泳分离活性序列，经过9轮筛选后，两个序列在文库中占主导地位。除了在一个序列中观察到单个A9U突变，而在另一个序列中观察到A9U和U7A的两个突变外，这两个序列大致符合共有核心区域。与共有序列的163μM-1min-1相比，这些核酶的催化效率分别为6.4和14.1μM-1min-1。有趣的是，在任何一个选择的序列中都没有发现共有序列。这种对共有序列的歧视归因于筛选过程中使用的酶的特异性。
