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School of Biomedical Sciences, Faculty of Medicine and Health Sciences, Queens Medical Centre, Nottingham NG7 2UH, UK. |
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School of Biomedical Sciences, Faculty of Medicine and Health Sciences, Queens Medical Centre, Nottingham NG7 2UH, UK. |
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| We have been interested in characterising the components of transcription pre inititiation complexes (PICs) on the promoters of mitogen responsive genes. These PICs contain general transcription factors, RNA polymerase II, specific transcription factors and other proteins involved in aspects of gene expression. Our experiments have focussed on promoters containing a serum response element (SRE), which binds SRF and the mitogen responsive ets factor Elk-1, and a serum inducible element (SIE), which recruits active STAT1 and STAT3 proteins. |
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We have harnessed protein mass spectrometry to address this issue. As described earlier, transcription complexes can be assembled in vitro on surrogate promoter templates to which a biotin moiety is attached. DNA protein complexes can then be isolated on streptavidin-coated magnetic beads (see Figure). Once isolated the activity of promoter complexes can be assayed in run-off transcription assays. Alternatively, the protein components can be eluted from the DNA, separated by SDS-PAGE and individual polypeptides can be then identified by a combination of peptide mass fingerprinting and de novo MS-MS sequencing. |
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The MS analyses have been performed by the Biopolymer Synthesis and Analysis Unit (BSAU), which is housed in the School of Biomedical Sciences. The Unit runs a micromass Q.Tof2, a micromass MALDI-TOF and uses the BioRad Proteome Works System to prepare samples for analysis. So far we have analysed data from SIE promoters bound by active STAT1 and STAT3. Over 200 proteins have been identified in these complexes. The data can be viewed with a bioinformatic tool accessed from the button below.
The unit's Q.Tof2 and Liquid Capilliary chromotography system |
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References
Drewett, V., Molina, H., Millar, A., Muller, S., von Hesler, F. and Shaw, P.E. (2001). DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos promoter and related sequences. Nucl. Acids. Res. 29, 479-487. (PDF)
Shaw, P.E., and Saxton, J. (2003) Ternary Complex Factors: Prime
Nuclear Targets For Mitogen-Activated Protein Kinases. Int. J. Biochem. Cell
Biol. 35, 1210-1226. (PDF)
