Schuppli D, Barrera I, Weber H
Institut für Molekularbiologie I Universität Zürich, Switzerland.
J Mol Biol. 1994 Nov 11;243(5):811-5. doi: 10.1006/jmbi.1994.1682.
In order to identify the structural elements important for the activity of the Q beta minus strand RNA as a template for Q beta replicase, a series of minus strand RNAs with internal or external deletions were prepared by in vitro transcription from suitable expression plasmids. The template activities of the deletion mutants were determined by single-round replication assays using purified replicase holoenzyme or core enzyme (lacking subunit alpha) in vitro. Two elements of RNA structure and/or sequence important for template activity were found. The first is a segment in the 5'-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previously recognized to be highly conserved in the small variant MDV-1 RNA and suggested to be involved in its template recognition. The second element is defined by two partially complementary sequence segments in the 3'-terminal region (map positions 557 to 576 and 24 to 35), that appear to be engaged in long-range base-pairing and may form the stem of a large secondary structure domain, whose branches are not necessary for template recognition. The results obtained with replicase holoenzyme and core enzyme were identical within the accuracy of the method. They confirm the absence of any role of S1 protein in the interaction of replicase with minus strand RNA and further emphasize the profound difference in the interactions of replicase with the plus and minus strand.
为了确定作为Qβ复制酶模板的Qβ负链RNA活性的重要结构元件，通过从合适的表达质粒进行体外转录，制备了一系列具有内部或外部缺失的负链RNA。缺失突变体的模板活性通过在体外使用纯化的复制酶全酶或核心酶（缺乏α亚基）的单轮复制测定来确定。发现了两个对模板活性重要的RNA结构和/或序列元件。第一个是5'末端区域的一个片段（图谱片段4078至4132），包含一个潜在的茎环结构，其序列先前被认为在小变体MDV-1 RNA中高度保守，并被认为参与其模板识别。第二个元件由3'末端区域的两个部分互补的序列片段定义（图谱位置557至576和24至35），它们似乎参与长程碱基配对，并可能形成一个大的二级结构域的茎，其分支对于模板识别不是必需的。在该方法的精度范围内，用复制酶全酶和核心酶获得的结果是相同的。它们证实了S1蛋白在复制酶与负链RNA相互作用中没有任何作用，并进一步强调了复制酶与正链和负链相互作用的深刻差异。
