Brown D, Gold L
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder 80309-0347, USA.
Biochemistry. 1995 Nov 14;34(45):14775-82. doi: 10.1021/bi00045a019.
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 data set of 54 replicatable 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.
通过复制/稀释方案，从两个随机序列RNA群体（一个长度为56个核苷酸，另一个为83个核苷酸）中分离出由Qβ复制酶复制的RNA。对选定的分子进行克隆和测序，生成了一个包含54个可复制RNA的数据集，这些RNA与Qβ复制酶的结合亲和力高于其从中选出的随机群体。对其中两个分子的缺失分析表明，RNA的内部区域负责Qβ复制酶的特异性结合。代表最小化RNA结合位点的截短分子似乎通过阻碍其与复制酶的结合来抑制全长分子的复制。这两个RNA的结合区域主要由连续的嘧啶组成。在数据集中85%的序列以及所有先前发表的可复制序列中都存在类似的富含C/U的区域。对其中一个可复制序列的多嘧啶结构域进行突变，使该分子与Qβ复制酶的亲和力降低了10倍，并完全消除了其被复制的能力。
