SELECTION AND CHARACTERIZATION OF RNAS REPLICATED BY Q-BETA REPLICASE
SELECTION AND CHARACTERIZATION OF RNAS REPLICATED BY Q-BETA REPLICASE
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DOI：
10.1021/bi00045a019
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发表时间：
1995-11-14
期刊：
影响因子：
2.9
通讯作者：
GOLD, L
中科院分区：
文献类型：
--
作者：
BROWN, D;GOLD, L
使用复制/稀释法从两个随机序列RNA群体(一个长度为56个核苷酸，第二个为83个核苷酸)中分离出由Qβ复制酶复制的RNA。选定的分子被克隆和测序，产生了54个可复制的RNA的数据集。对四个主导各自选择池的分子进行了表征。所有四个RNA与Qβ复制酶结合的亲和力都高于从中选择它们的随机群体。对其中两个分子的缺失分析表明，RNA的内部区域是。负责Qβ复制酶的特异性结合。代表最小RNA结合位点的截短分子可以抑制全长分子的复制，显然是通过阻止它们与复制酶结合来实现的。这两个RNA的结合区主要由延长的嘧啶类化合物组成。相似的富C/U区存在于数据集中85%的序列以及所有先前发表的可复制序列中。其中一个可复制序列的多嘧啶结构域的突变使该分子对Qβ复制酶的亲和力降低了10倍，并完全丧失了其复制能力。
RNAs replicated by Q beta replicase were isolated from two random sequence RNA populations (one 56 nucleotides in length, the second 83) using a replication/dilution protocol. The selected molecules were cloned and sequenced, generating a data set of 54 replicatable RNAs. Four molecules that dominated their respective selection pools were characterized. All four of the RNAs bound with higher affinity to Q beta replicase than did the random populations from which they were selected. Deletion analyses on two of the molecules indicated that internal regions of the RNAs were. responsible for the specific binding of Q beta replicase. Truncated molecules representing the minimized RNA binding sites could inhibit replication of the full-length molecules, apparently by obstructing their binding to the replicase. The binding regions of the two RNAs were dominated by extended runs of pyrimidines. Similar C/U-rich regions existed in 85% of the sequences in the data set as well as in all of the previously published replicatable sequences. Mutation of the polypyrimidine domain of one of the replicatable sequences reduced the affinity of the molecule for Q beta replicase by 10-fold and completely abolished its ability to be replicated.