BACKGROUND: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ-F1 or its silkworm counterpart BmFTZ-F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ-F1 (or BmFTZ- F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription. RESULTS: To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ-F1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acid sequence of the factor showed no homology with proteins in the databases. Farwestern analyses and glutathione S-transferase interaction assays demonstrated that MBF2 makes a direct contact with the beta-subunit of TFIIA. In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires the MBF2-TFIIA interaction. When recombinant MBF2 was added to the HeLa cell nuclear extract in the presence of MBF1 and FTZ622 bearing the DNA-binding region of FTZ-F1, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the MBF2-TFIIA interaction. CONCLUSION: MBF2 activates transcription through its interaction with TFIIA. Selective transcriptional activation occurs when MBF2 is recruited to a promoter carrying the FTZ-F1 binding site by FTZ-F1 and MBF1.
BACKGROUND: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ-F1 or its silkworm counterpart BmFTZ-F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ-F1 (or BmFTZ- F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription. RESULTS: To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ-F1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acid sequence of the factor showed no homology with proteins in the databases. Farwestern analyses and glutathione S-transferase interaction assays demonstrated that MBF2 makes a direct contact with the beta-subunit of TFIIA. In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires the MBF2-TFIIA interaction. When recombinant MBF2 was added to the HeLa cell nuclear extract in the presence of MBF1 and FTZ622 bearing the DNA-binding region of FTZ-F1, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the MBF2-TFIIA interaction. CONCLUSION:MBF2 activates transcription through its interaction with TFIIA. Selective transcriptional activation occurs when MBF2 is recruited to a promoter carrying the FTZ-F1 binding site by FTZ-F1 and MBF1.