Investigation of molecular size of transcription factor TFIIE in solution.
Human 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 TFIIEalpha (50 kDa) and TFIIEbeta (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 alphabeta 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 alphabeta 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.
Itoh
Yoshiyuki
Y
Graduate School of Integrated Science, Yokohama City University, Kanagawa, Japan.
Unzai
Satoru
S
Sato
Mamoru
M
Nagadoi
Aritaka
A
Okuda
Masahiko
M
Nishimura
Yoshifumi
Y
Akashi
Satoko
S
D020816:Amino Acid Motifs; D002850:Chromatography, Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012319:RNA Polymerase II / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012996:Solutions / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019473:Transcription Factors, TFII / Q000737:chemistry*; D014461:Ultracentrifugation
D011993:Recombinant Fusion Proteins; D012996:Solutions; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D012319:RNA Polymerase II