RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits
RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits
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DOI：
10.1128/mcb.24.16.7043-7058.2004
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发表时间：
2004-08-01
影响因子：
5.3
通讯作者：
Coulombe, B
中科院分区：
文献类型：
--
作者：
Jeronimo, C;Langelier, MF;Coulombe, B
我们已经编程人类细胞表达生理水平的重组RNA聚合酶II（RNAPII）亚基携带串联亲和纯化（TAP）标签。双亲和层析允许简单而有效地分离一个复杂的包含所有12个RNAPII亚基，一般转录因子TFIIB和TFIIF，RNAPII磷酸酶Fcp1，和一个新的153 kDa的多肽未知的功能，我们命名为RNAPII相关蛋白I（RPAPI）。TAP标记的RNAPII复合物在体外和体内都具有功能活性。RPAPI在RNAPII转录中的作用是通过关闭Ydr527wp的合成来建立的，Ydr527wp是与RPAPI同源的酿酒酵母蛋白，并证明了全局基因表达的变化与酵母RNAPII亚基RpbII的丢失所引起的变化相似。我们还使用了TAP标记的Rpb 2，其中叉环1和开关3中有突变，这两个结构元件战略性地位于活性中心内，以开始解决这些元件在转录反应期间酶与模板DNA相互作用中的作用。
We have programmed human cells to express physiological levels of recombinant RNA pollymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein I (RPAPI). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAPI in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAPI, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit RpbII. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.