Gross S, Moore C
Department of Molecular Biology and Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Stearns 509, 136 Harrison Avenue, Boston, MA 02111, USA.
Proc Natl Acad Sci U S A. 2001 May 22;98(11):6080-5. doi: 10.1073/pnas.101046598. Epub 2001 May 8.
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.
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，许多蛋白质在切割和/或聚腺苷酸化过程中发挥作用。通过从单独表达和纯化的蛋白质中重组活性CF I，我们发现CF I包含五个亚基，即Rna14、Rna15、Pcf11、Clp1和Hrp1。当与CF II混合时，这五个亚基对于在体外重组切割活性是必需且充分的；当与聚腺苷酸化因子I、纯化的聚（A）聚合酶和聚（A）结合蛋白混合时，它们对于特异性聚腺苷酸化也是必需且充分的。对单个蛋白质-蛋白质相互作用的分析支持了CF I的一种结构模型，其中Pcf11同时与Rna14、Rna15和Clp1相互作用，而Rna14则连接Rna15和Hrp1。
