Reference: Mischerikow N, et al. (2009) In-depth profiling of post-translational modifications on the related transcription factor complexes TFIID and SAGA. J Proteome Res 8(11):5020-30

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Abstract


The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multi-pronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate < 1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive pre-fractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.

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Journal Article
Authors
Mischerikow N, Spedale G, Altelaar M, Timmers HT, Pijnappel P, Heck AJ
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