PDF(Pubmed)
Abstract：
The 3\' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.
摘要：
几乎所有真核mRNAs的3'末端都是在必需的两步加工反应中产生的：延伸前体的内切核酸裂解，然后添加poly（A）尾。通过从过度生产和纯化的蛋白质中重建反应，我们提供了对RNA加工必需的14种多肽和两种具有刺激性的多肽的最少列表。在取决于聚腺苷酸化信号AAUAAA的反应中，重建系统在对应于体内使用的单个优选位点切割pre-mRNA。在蛋白质中，裂解因子I刺激裂解，但不是必需的，与其在替代聚腺苷酸化中的突出作用一致。RBBP6是必需的，结构数据显示它接触并可能通过其DWNN结构域激活核酸内切酶CPSF73。RNA聚合酶II的C末端结构域是可有可无的。ATP,但不是它的水解，通过以亚微摩尔亲和力与裂解因子II的hClp1亚基结合来支持RNA裂解。
