Structural characterization of human general transcription factor TFIIF in solution
Structural characterization of human general transcription factor TFIIF in solution
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DOI：
10.1110/ps.073258108
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发表时间：
2008-03-01
期刊：
影响因子：
8
通讯作者：
Nishimura, Yoshifumi
中科院分区：
文献类型：
--
作者：
Akashi, Satoko;Nagakura, Shinjiro;Nishimura, Yoshifumi
人通用转录因子IIF（TFIIF）是与RNA聚合酶II（Pol II）相关的转录前起始复合物（PIC）的一个组分，其特征在于尺寸排阻色谱（SEC）、电喷雾电离质谱（ESI-MS）和化学交联。由等摩尔比的α和β亚基组成的重组TFIIF经细菌表达、纯化至均一，并发现其在人体外转录系统中具有与天然TFIIF相似的转录活性。如先前报道的，纯化的TFIIF的SEC表明该蛋白具有> 200 kDa的大小。相比之下，纯化样品的ESI-MS得到87 kDa的分子大小，表明TFIIF是ab异二聚体，这通过交联TFIIF组分的基质辅助激光解吸/电离（MALDI）MS证实。最近的电子显微镜（EM）和光交联研究表明，酵母TFIIF同源物含有Tfg 1和Tfg 2，对应于人的a和B亚基，在PIC中以异源二聚体存在，因此人TFIIF也可能以异源二聚体存在，即使在PIC中。在酵母PIC中，EM和光交联研究显示了TFIIE和TFIIF沿DNA沿着相互定位的不同结果。我们已经通过ESI-MS、SEC和化学交联研究了人TFIIF和TFIIE之间的直接相互作用;然而，至少在溶液中没有观察到直接相互作用。这与之前的光交联观察一致，即TFIIF和TFIIE分别在酵母PIC中Pol II中央裂缝的两侧上侧翼DNA。
Human general transcription factor IIF ( TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography ( SEC), electrospray ionization mass spectrometry ( ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of alpha and beta subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size > 200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an ab heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy ( EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human a and b subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC.