Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase
Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase
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DOI：
10.1074/jbc.273.12.6724
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发表时间：
1998-03-20
影响因子：
4.8
通讯作者：
Kirkegaard, K
中科院分区：
文献类型：
--
作者：
Beckman, MTL;Kirkegaard, K
脊髓灰质炎病毒 RNA 依赖性 RNA 聚合酶与单链 RNA 协同结合。我们使用长度增加的寡核苷酸进行结合滴定，确定了脊髓灰质炎病毒聚合酶的最小RNA结合位点大小。当oligo(U)的长度从8个核苷酸(nt)增加到10个核苷酸(nt)时，观察到亲和力显着增加，认为聚合酶结合的RNA的最小大小是10nt。当oligo(U)达到24nt时，亲和力再次增加，这表明24个核苷酸的RNA可以被两个聚合酶分子占据。野生型聚合酶与oligo(U)(12)和oligo(U)(24) RNA的直接结合显示出与该模型一致的亲和力和协同性差异。 oligo(U)(10) 结合亲和力的增加表明，RNA 结合决定簇在聚合酶结构上间隔很宽，或者聚合酶在填充其 RNA 结合位点时发生了显着的构象变化。
The poliovirus RNA-dependent RNA polymerase binds cooperatively to single stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wildtype polymerase to oligo(U)(12) and oligo(U)(24) RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)(10) suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA binding site.