Molecular flexibility and discontinuous translocation of a non-templated polymerase.
Molecular flexibility and discontinuous translocation of a non-templated polymerase.
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非模板聚合酶的分子灵活性和不连续易位。
DOI：
10.1016/j.jmb.2004.01.058
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Gershon,PD
中科院分区：
文献类型：
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作者：
Johnson,L;Liu,S;Gershon,PD
关于非模板核酸聚合酶相对于单链引物的易位，人们知之甚少。痘苗病毒聚(A)聚合酶VP55是一种易位病毒，它在只有三个核苷残基(作为(RU)2-N15-RU基序)的引物中加入∼3-7腺苷酸尾，在U含量较高的引物中加入∼25-30腺苷酸尾。在这里，VP55相对于其引物的易位有三种模型，即：(A)刚性蛋白质/刚性核酸；(B)柔性蛋白质/刚性核酸；(C)刚性蛋白质/柔性核酸。对游离和共价连接的VP55引物的分析支持(B)或(C)含有被动空间阻断的版本，并提出聚合酶和引物之间存在两个相对运动区域。在3‘端加入富含6个核苷酸的尿苷酸补丁，在不影响初始结合亲和力的情况下，将聚合酶从∼3-7个核苷酸增加到∼25-30个核苷酸的尾部。通过将该斑块合成为(RU/DC)池，可以检测到不连续的聚合酶运动。
Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a ∼3–7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)2–N15–rU motif), and a ∼25–30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3′ end switched the polymerase from ∼3–7nt to ∼25–30nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.