Whoriskey S K, Usman N, Szostak J W
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2465-9. doi: 10.1073/pnas.92.7.2465.
We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The ribozyme is derived from a group I intron and consists of three RNA fragments of 36, 43, and 59 nt that self-assemble to form a catalytically active complex. We have site-specifically substituted ribonucleotide analogs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity. In contrast, neither the 2'-hydroxyl nor the exocyclic amino group of the conserved guanosine in the guanosine binding site is necessary for catalysis. By allowing the ribozyme to be modified as easily as its substrates, this synthetic ribozyme system should be useful for testing specific hypotheses concerning ribozyme-substrate interactions and tertiary interactions within the ribozyme.
我们描述了一种催化模板导向的寡核苷酸连接的核酶的完全化学合成。合成核酶的比活性与用T7 RNA聚合酶体外转录产生的相同酶的比活性几乎相同。该核酶源自I组内含子,由36、43和59个核苷酸的三个RNA片段组成,这些片段自组装形成催化活性复合物。我们已将核糖核苷酸类似物位点特异性地取代到该酶中,并鉴定出两个对完全催化活性必需的2'-羟基。相比之下,鸟苷结合位点中保守鸟苷的2'-羟基和环外氨基对于催化都不是必需的。通过使核酶能够像其底物一样容易地被修饰,这种合成核酶系统应该有助于测试关于核酶-底物相互作用以及核酶内三级相互作用的特定假设。