HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: ps.073258108v1 17/3/389    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Akashi, S. Articles by Nishimura, Y. Search for Related Content PubMed PubMed Citation Articles by Akashi, S. Articles by Nishimura, Y. Social Bookmarking                   What's this?

Structural characterization of human general transcription factor TFIIF in solution

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 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 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 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

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073258108.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?