Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates
Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates
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DOI：
10.1093/emboj/18.13.3767
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发表时间：
1999-07-01
期刊：
影响因子：
11.4
通讯作者：
Ortín, J
中科院分区：
文献类型：
--
作者：
González, S;Ortín, J
流感病毒RNA聚合酶是由PB1、PB2和PA亚基组成的异源三聚体。PB1是该复合物的核心，并决定了聚合酶的活性。我们通过体外结合和西北分析研究了PB1与模型cRNA模板的相互作用。与模型cRNA的结合是特异性的，其表观K-d近似于7 x 10(-8) m。与与vRNA的相互作用相反，PB1能够同时结合cRNA长柄的5‘和3’臂。PB1的N端139个氨基酸和267 ~ 493个位置之间的序列证明与cRNA结合呈阳性，而与vRNA模板的相互作用先前被定位在N端和c端区域。利用vRNA或cRNA长柄的5‘和3’臂进行竞争实验，结果表明两个模板共享n端结合位点，数据表明PB1 rna结合位点由：(i)位于n端的残基（可能在vRNA和cRNA结合中常见），以及（ii）来自PB1中心部分的残基（对于cRNA）或（iii）来自PB1 c端区域的残基（对于vRNA），这表明PB1在与cRNA和vRNA模板结合时经历了构象变化。
The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent K-d of similar to 7 x 10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates, The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates.