Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I
Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I
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DOI：
10.1073/pnas.101046598
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发表时间：
2001-05-22
影响因子：
11.1
通讯作者：
Moore, C
中科院分区：
文献类型：
--
作者：
Gross, S;Moore, C
酿酒酵母中 mRNA 3' 末端的切割和聚腺苷酸化需要多种因子，其中之一是切割因子 I (CF I)。从酵母提取物中纯化 CF I 活性表明许多蛋白质在裂解和/或多聚腺苷酸化中发挥作用。通过从单独表达和纯化的蛋白质中重建活性 CF I，我们发现 CF I 包含五个亚基：Rna14、Rna15、Pcf11、Clp1 和 Hrp1。当与 CF II 混合时，这五种对于在体外重建裂解活性以及与聚腺苷酸化因子 I、纯化的聚腺苷酸聚合酶和聚腺苷酸结合蛋白混合时的特异性聚腺苷酸化是必要且充分的。对单个蛋白质-蛋白质相互作用的分析支持 CF I 的结构模型，其中 Pcf11 同时与 Rna14、Rna15 和 Clp1 相互作用，而 Rna14 桥接 Rna15 和 Hrp1。
Cleavage and polyadenylation of mRNA 3 ' ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein-protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.