Investigation of molecular size of transcription factor TFIIE in solution
Investigation of molecular size of transcription factor TFIIE in solution
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DOI：
10.1002/prot.20647
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发表时间：
2005-11-15
影响因子：
2.9
通讯作者：
Akashi, S
中科院分区：
文献类型：
--
作者：
Itoh, Y;Unzai, S;Akashi, S
人通用转录因子HE（TFIIE），与RNA聚合酶II相关的转录前起始复合物的一个组成部分，其特征在于尺寸排阻色谱，质谱，分析超离心，和小角X射线散射（SAXS）。将重组人TFIIE纯化至同质，并通过SDS-PAGE显示含有等摩尔量的TFIIE α（50 kDa）和TFIIE β（35 kDa）。在纯化样品的尺寸排阻色谱分析中，如已经报道的，TFIIE显示为170-kDa α（2）β（2）异四聚体。然而，通过使用电喷雾电离质谱法，纯化的样品给出84，152 +/- 5的分子量，表明TFIIE是上异源二聚体而不是异源四聚体。TFIIE的分析超离心实验表明，只有分子量约为1000的单一组分，才能被分离出来。80，000存在于溶液中，这也表明了异二聚体。此外，它的异常棒状分子形状被SAXS证实。TFIIE的棒状分子形状可能在尺寸排阻色谱中误导了较大的分子尺寸，该尺寸排阻色谱是通过球状蛋白校准的。它表明，TFIIE存在的异二聚体在我们目前的条件下，在溶液中，虽然两个分子的异二聚体可能需要与RNA聚合酶II启动转录过程的preinitiation复合物的形成。Proteins 2005;61：633-641. (c)2005 Wiley-Liss，Inc.
Human general transcription factor HE (TFIIE), a component of a transcription preinitiation complex associated with RNA polymerase II, was characterized by size-exclusion chromatography, mass spectrometry, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Recombinant human TFIIE was purified to homogeneity and shown to contain equimolar amounts of TFIIE alpha (50 kDa) and TFIIE beta (35 kDa) by SDS-PAGE. In the analysis of size-exclusion chromatography of the purified sample, as already reported, TFIIE was shown to be a 170-kDa alpha(2)beta(2) heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 +/- 5, indicating that TFIIE is an up heterodimer but not a heterotetramer. Analytical ultracentrifugation experiment of TFIIE provided that only a single component with the molecular mass of ca. 80,000 existed in solution, also suggesting an up heterodimer. In addition, its extraordinarily rod-like molecular shape was confirmed by SAXS. It is likely that the rod-like molecular shape of TFIIE has misled larger molecular size in size-exclusion chromatography, which was calibrated by globular proteins. It is demonstrated that TFIIE exists as a heterodimer under our present conditions in solution, although two molecules of heterodimer might be required for the formation of the preinitiation complex with RNA polymerase II for starting the transcription process. Proteins 2005;61:633-641. (c) 2005 Wiley-Liss, Inc.