SATOKO AKASHI1, SHINJIRO NAGAKURA1, SEIJI YAMAMOTO2, MASAHIKO OKUDA1, YOSHIAKI OHKUMA2,3, AND YOSHIFUMI NISHIMURA1
- International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan
- Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
- Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
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.
CovalX Technology Used
Recombinant human TFIIF was obtained through co-expression using a plasmid. The sample was then cross-linked using the CovalX K200 Stabilization Kit. TFIIF (27 μM, 13.5 μM or 3 μM) in Buffer A (20 mM sodium phosphate (pH 7.9), 500 mM NaCl, 10 mM 2-mercaptoethanol, 10% glycerol) was mixed with the K200 using a ratio of 9:1 by volume. The samples were then incubated for 1 hour at room temperature before being mixed with matrix (10 mg/ml sinapic acid in 0.1% TFA/50% acetonitrile aqueous solution). 1 μL of the sample/matrix mixture was spotted on a stainless steel MALDI sample plate before being dried at room temperature. 1 μL of cold 0.1% TFA was spotted on top of the dried sample spot to desalt the sample and then immediately absorbed back by the pipette before being discarded. 0.5 μL of matrix solution aliquot was added to the spot and dried before the plate was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.