Structural characterization of human general transcription factor TFIIF in solution.
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 alphabeta 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 alpha and beta 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.
Akashi
Satoko
S
International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan.
Nagakura
Shinjiro
S
Yamamoto
Seiji
S
Okuda
Masahiko
M
Ohkuma
Yoshiaki
Y
Nishimura
Yoshifumi
Y
D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D019281:Dimerization; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D012996:Solutions; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; C056604:transcription factor TFIIF