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Structural characterization of human general transcription factor TFIIF in solution
Author(s) -
Akashi Satoko,
Nagakura Shinjiro,
Yamamoto Seiji,
Okuda Masahiko,
Ohkuma Yoshiaki,
Nishimura Yoshifumi
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073258108
Subject(s) - transcription factor ii f , transcription factor , transcription factor ii b , computational biology , genetics , biology , chemistry , promoter , gene , transcriptional regulation , gene expression
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 α and β 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 αβ 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 α and β 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.