Site-specific cross-linking of TBP in vivo and in vitro reveals a direct functional interaction with the SAGA subunit Spt3.
The TATA-binding protein (TBP) is critical for transcription by all three nuclear RNA polymerases. In order to identify factors that interact with TBP, the nonnatural photoreactive amino acid rho-benzoyl-phenylalanine (BPA) was substituted onto the surface of Saccharomyces cerevisiae TBP in vivo. Cross-linking of these TBP derivatives in isolated transcription preinitiation complexes or in living cells reveals physical interactions of TBP with transcriptional coregulator subunits and with the general transcription factor TFIIA. Importantly, the results show a direct interaction between TBP and the SAGA coactivator subunits Spt3 and Spt8. Mutations on the Spt3-interacting surface of TBP significantly reduce the interaction of TBP with SAGA, show a corresponding decrease in transcription activation, and fail to recruit TBP to a SAGA-dependent promoter, demonstrating that the direct interaction of these factors is important for activated transcription. These results prove a key prediction of the model for stimulation of transcription at SAGA-dependent genes via Spt3. Our cross-linking data also significantly extend the known surfaces of TBP that directly interact with the transcriptional regulator Mot1 and the general transcription factor TFIIA.
Mohibullah
Neeman
N
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Hahn
Steven
S
D000251:Adenosine Triphosphatases; D003432:Cross-Linking Reagents / Q000737:chemistry; D004265:DNA Helicases / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D035381:TATA-Binding Protein Associated Factors / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D035561:Transcription Factor TFIIA / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D003432:Cross-Linking Reagents; C507785:SAGA complex, S cerevisiae; C075582:SPT3 protein, S cerevisiae; C089445:SPT8 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D010649:Phenylalanine; D000251:Adenosine Triphosphatases; C073676:MOT1 protein, S cerevisiae; D004265:DNA Helicases