DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences.
Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional 'transcriptosome' has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.
Drewett
V
V
School of Biomedical Sciences, D Floor, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Molina
H
H
Millar
A
A
Muller
S
S
von Hesler
F
F
Shaw
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008863:Microspheres; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D026362:Serum Response Factor; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D050807:ets-Domain Protein Elk-1
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D026362:Serum Response Factor; D014157:Transcription Factors; C066114:biotin-streptavidin complex; D050807:ets-Domain Protein Elk-1; D001710:Biotin; D004247:DNA