Abstract
The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (Kd ∼1.0–10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (∼1200 Å2). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.
Highlights
In eukaryotes the regulation of transcription initiation involves coordinated interactions between a large number of proteins and complexes, including components of the basal transcription machinery, sequence-specific DNA-binding transcription factors such as B-Myb, coactivators and corepressors
Spectroscopic Characterisation of the B-Myb transactivation domain (TAD) A typical far-UV CD spectrum obtained for the B-Myb TAD is shown in figure 2A and is characterised by a large negative peak at approximately 200 nm, which together with the overall profile of the spectrum strongly suggests that the isolated B-Myb TAD forms a random coil polypeptide, with very little if any regular secondary structure
The intrinsic fluorescence spectrum of B-Myb TAD (figure 2B (i)) is typical of that expected for an Conservative substitutions are highlighted in an open box and non-conservative highlighted in grey
Summary
In eukaryotes the regulation of transcription initiation involves coordinated interactions between a large number of proteins and complexes, including components of the basal transcription machinery, sequence-specific DNA-binding transcription factors such as B-Myb, coactivators and corepressors. A number of B-Myb regulated genes have been identified in which activation of transcription involves the binding of B-Myb to consensus target sites (MBS) in their promoter or enhancer regions, leading to the recruitment of essential partner proteins such as the coactivators p300 and CBP [11], [17], [19], [20], [21], [22], [23], [24], [25], [26]. The precise molecular basis of the interaction and functional synergy between B-Myb and p300 remains to be determined and is the focus of the work reported here In this communication we report detailed characterisation of the principal domains involved in B-Mybp300 interactions and of the complex formed, including identification of the binding surface for the B-Myb TAD on the TAZ2 domain of p300
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