Novel subunits of the TATA binding protein free TAFII-containing transcription complex identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry following one-dimensional gel electrophoresis.
Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require the transcription factor II D (TF(II)D), a complex comprising the TATA binding protein (TBP) and TBP-associated factors. However, another multiprotein complex isolated more recently and called TFTC (TBP-free TAF(II )containing complex), was shown to mediate initiation of RNA polymerase II (Pol II) transcription in the absence of TF(II)D as well as specific acetylation of histone H3 in a nucleosomal context. Several subunits of the TFTC complex were already identified using classical methods such as Edman based microsequencing and Western blot analysis. In this article we present a mass spectrometry based proteomic approach to confirm previous results and to identify other possible subunits of the TFTC complex. The TFTC complex was separated on one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis and analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting. Identifications were realized after databank searches. This new characterization of TFTC complex confirmed the presence of already described subunits (TRRAP, GCN5, SAP130/KIA0017, TAF(II)150, TAF(II)135, TAF(II)100, TAF(II)80, TAF(II)20, SPT3 and PAF65beta). Moreover, a good coverage of these sequences was obtained. Interestingly, TAF(II)32 and PAF6alpha were also determined as potential novel subunits of TFTC. These results together show the suitability and the great potential of this method and offer new perspectives in fundamental studies of transcription factor complexes.
Cavusoglu
Nükhet
N
Laboratoire de Spectrométrie de Masse Bio-Organique, UMR 7509, Strasbourg, France.
Brand
Marjorie
M
Tora
Laszlo
L
Van Dorsselaer
Alain
A
D015153:Blotting, Western; D019992:Databases as Topic; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D035181:TATA-Box Binding Protein / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000378:metabolism
D006657:Histones; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D019473:Transcription Factors, TFII