COLIPHAGE Q-BETA RNA REPLICATION - RNA CATALYTIC FOR SINGLE-STRAND RELEASE
COLIPHAGE Q-BETA RNA REPLICATION - RNA CATALYTIC FOR SINGLE-STRAND RELEASE
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DOI：
10.1016/0042-6822(91)90430-j
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发表时间：
1991-10-01
期刊：
影响因子：
3.7
通讯作者：
MILLS, DR
中科院分区：
文献类型：
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作者：
AXELROD, VD;BROWN, E;MILLS, DR
我们已经为O-β复制酶构建了14个重组模板，每个模板都有一个外源反向重复序列或一个没有重复序列。这些模板被用来通过Qβ复制酶启动体外复制，其数量达到了酶的饱和。我们观察到，在重组序列中假定包含二级结构的RNA的复制率从野生型MDV-1 RNA对照的33%到69%不等，无论插入的发夹的大小如何。此外，大多数新合成的RNA是以单链形式存在的。或者，含有预期不会形成二级结构的外源序列的RNA的复制率低于MDV-1的25%。在每种情况下，反应速度与插入的长度相关，并且大多数产物RNA由双链分子(互补的正链和负链杂交在一起)组成。当将这些相同的重组RNA用于RNA模板摩尔量比复制酶低106-107倍的反应时，只有那些假定含有二级结构的RNA在复制反应中存活。我们的结果与Qβ复制酶合成过程中发夹结构的形成直接影响单链RNA产物再生的理论一致。
We have generated 14 recombinant RNA templates for Oβ replicase, each having either an exogenous inverted repeat sequence or a sequence with no repeat. These templates were used to initiatein vitroreplication by Qβ replicase in amounts that saturated the enzyme. We observed that replication rates for RNAs that putatively contained secondary structures in the recombinant sequences ranged from 33 to 69% that of a wild-type MDV-1 RNA control, regardless of the size of the inserted hairpin. Moreover, most of the newly synthesized RNA was present as single strands. Alternatively, RNAs that contained exogenous sequences not expected to form secondary structures exhibited replication rates less than 25% that of MDV-1. In each case, the reaction rate was correlated with the length of the insertion, and the majority of product RNA consisted of duplexed molecules (complementary plus and minus strands hybridized together). When these same recombinant RNAs were used in reactions in which the molar amount of RNA template was 106–107 times lower than that of the replicase, only those that putatively contained secondary structures survived in the replication reaction. Our results are consistent with the theory that hairpin structure formation during RNA synthesis by Qβ replicase directly influences the regeneration of single-stranded RNA products.