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Published online before print January 24, 2008, 10.1110/ps.073258108
Protein Science (2008), 17:389-400. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
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Structural characterization of human general transcription factor TFIIF in solution

Satoko Akashi1, Shinjiro Nagakura1, Seiji Yamamoto2, Masahiko Okuda1, Yoshiaki Ohkuma2,3, and Yoshifumi Nishimura1

1 International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan
2 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
3 Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan

(RECEIVED September 20, 2007; FINAL REVISION November 16, 2007; ACCEPTED November 27, 2007)

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 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 {alpha}β 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 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.

Keywords: TFIIF; size-exclusion chromatography (SEC); electrospray ionization mass spectrometry (ESI-MS); chemical cross-linking; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS); TFIIE; heterodimer


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