Hannan R D, Hempel W M, Cavanaugh A, Arino T, Dimitrov S I, Moss T, Rothblum L
Henry Hood Research Program, Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2618, USA.
J Biol Chem. 1998 Jan 9;273(2):1257-67. doi: 10.1074/jbc.273.2.1257.
Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library. Analyses of the deduced amino acid sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the beta' and beta subunits of Escherichia coli RNA polymerase I. Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin. N1S1 cell lines expressing recombinant His-tagged A194 and FLAG-tagged A127 proteins were isolated. These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column. Immunoprecipitation of A194 from 32P metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein. Incubation of the FLAG affinity-purified RNA polymerase I complex with [gamma-32P]ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s). One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.
从鲁伯肝癌cDNA文库中分离出编码大鼠RNA聚合酶I两个最大亚基（命名为A194和A127）的重叠cDNA克隆。对推导的氨基酸序列分析表明，A194和A127是酵母A190和A135的同源物，并且与大肠杆菌RNA聚合酶I的β'和β亚基具有同源性。针对重组A194和A127蛋白产生的抗体在源自哺乳动物的总细胞蛋白的蛋白质印迹上识别出约190 kDa和120 kDa的单一蛋白。分离出表达重组His标签的A194和FLAG标签的A127蛋白的N1S1细胞系。这些蛋白被整合到功能性RNA聚合酶I复合物中，并且含有FLAG标签的A127的活性酶可以在抗FLAG亲和柱上通过单一色谱步骤免疫纯化至约80%的纯度。用抗A194抗血清从经32P代谢标记的细胞中免疫沉淀A194，表明该亚基是一种磷蛋白。将FLAG亲和纯化的RNA聚合酶I复合物与[γ-32P]ATP一起温育导致RPI的A194亚基发生自磷酸化，表明存在相关的激酶。其中一种激酶被证明是CK2，一种与细胞生长和增殖调节有关的丝氨酸/苏氨酸蛋白激酶。
