AbstractHuman general transcription factor IIE (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 TFIIEα (50 kDa) and TFIIEβ (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 α2β2 heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 ± 5, indicating that TFIIE is an αβ 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 αβ 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. © 2005 Wiley‐Liss, Inc.