Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex.
The basal factor TFIIA has been shown to act early during initiation in both the mammalian and yeast transcription systems, but a TFIIA-like activity has not been identified in Drosophila. While characterizing the Drosophila TFIID complex, we discovered that a 30-kD protein that cofractionated with dTFIID was homologous to the previously identified, large subunit of yeast TFIIA. Here, we report the cloning and biochemical characterization of Drosophila TFIIA-L. Coimmunoprecipitation studies with anti-dTBP, anti-dTFIIA-L, and anti-TAF antibodies indicated a tight association of the endogenous dTFIIA and dTFIID. However, dTFIIA could be dissociated from dTFIID under conditions that did not elute the TAFs, and the eluted material had mobility shift and transcriptional activities associated with TFIIA. Peptide sequence and Western analysis with antibodies raised against the amino- and carboxy-terminal portions of recombinant dTFIIA-L revealed that a precursor 48-kD species was cleaved in vivo, giving rise to the 30- and 20-kD subunits of dTFIIA that remain associated with each other and with dTFIID. Protein-protein interaction assays identified dTBP and dTAFII110 as targets for binding TFIIA in the TFIID complex. These results suggest that TFIIA may form a specific complex with both TAFs and other components of the transcriptional machinery during formation of the initiation complex.
Yokomori
K
K
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Admon
A
A
Goodrich
J A
JA
Chen
J L
JL
Tjian
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007301:Insect Hormones / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D007301:Insect Hormones; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; C084263:TfIIA-L protein, Drosophila; D035561:Transcription Factor TFIIA; D014157:Transcription Factors