SEQUENCE-ANALYSIS OF RNA SPECIES SYNTHESIZED BY Q-BETA REPLICASE WITHOUT TEMPLATE
SEQUENCE-ANALYSIS OF RNA SPECIES SYNTHESIZED BY Q-BETA REPLICASE WITHOUT TEMPLATE
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DOI：
10.1021/bi00069a021
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发表时间：
1993-05-11
期刊：
影响因子：
2.9
通讯作者：
LUCE, R
中科院分区：
文献类型：
--
作者：
BIEBRICHER, CK;LUCE, R
Qbeta复制酶以高效率自催化方式扩增某些短链RNA模板。在不存在不均匀添加的模板的情况下，在高酶和底物浓度的条件下以及在长滞后时间之后观察到通过Qbeta复制酶合成新的RNA种类。即使在相同的条件下，不同的实验也会产生不同的RNA种类。几个独立的无模板产品的序列已被确定通过克隆它们的cDNA到质粒中的一种新的克隆程序。它们的核苷酸链长度很小，范围从25到约50个核苷酸。虽然它们的一级序列除了不变的5 '-末端G和3'-末端C簇外是不相关的，但它们的初步二级结构显示了一个共同的原则，即它们的正链和负链在5'末端都有一个茎，而3'末端是未配对的。通过Qbeta复制酶直接积累足够量的早期无模板合成产物被合成过程固有的不可再现性和在进化过程扩增期间产物的快速变化所阻止，但是大量的这种RNA可以通过从cDNA克隆转录而在体外合成。在无模板反应中产生的RNA种类复制比先前表征的优化的RNA种类慢得多。这些实验结果说明了生物信息是如何通过反复试验以小比特的形式获得的。
Qbeta replicase amplifies certain short-chained RNA templates autocatalytically with high efficiency. In the absence of extraneously added template, synthesis of new RNA species by Qbeta replicase is observed under conditions of high enzyme and substrate concentrations and after long lag times. Even under identical conditions, different RNA species are produced in different experiments. The sequences of several independent template-free products have been determined by cloning their cDNAs into plasmids by a novel cloning procedure. Their nucleotide chain lengths are small, ranging from 25 to about 50 nucleotides. While their primary sequences are unrelated except for the invariant 5'-terminal G and 3'-terminal C clusters, their tentative secondary structures show a common principle both their plus and minus strands have a stem at the 5' terminus, while the 3' terminus is unpaired. Direct accumulation of sufficient quantities of early template-free synthesis products by Qbeta replicase is prevented by the inherent irreproducibility of the synthesis process and by the rapid change of the products during amplification by evolution processes, but large amounts of such RNA can be synthesized in vitro by trancription from the cDNA clones. RNA species produced in template-free reactions replicate much more slowly than the optimized RNA species characterized previously. These experimental results illustrate how biological information can be gained in small bits by trial and error.