STRUCTURE AND FUNCTION OF THE SMALL-SUBUNIT OF TFIIF (RAP30) FROM DROSOPHILA-MELANOGASTER

To study the mechanism of basal transcription by RNA polymerase II, a cDNA encoding the Drosophila homologue of the small subunit of TFIIF ( also referred to as TFIIF30, RAP30, factor 5b, and gamma) was isolated . The Drosophila TFIIF30 gene is located at region 86C on the right ar m of the third chromosome. The protein encoded by the cDNA, termed dTF IIF30, was synthesized in Escherichia coli and purified to greater tha n 95% homogeneity. In reconstituted transcription reactions with purif ied basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, d esignated dF30(119-276), which contains the carboxyl-terminal 158 amin o acid residues of dTFIIF30, was found to possess approximately 50% of the transcriptional activity as full-length dTFIIF30. The interaction of dTFIIF30 with the large subunit of TFIIF (also referred to as TFII F74, RAP74, factor 5a, and beta) was investigated by glycerol gradient sedimentation analyses. In these experiments, dTFIIF30, but not dF30( 119-276), assembled into a stable heteromeric complex with TFIIF74. Th ese results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxyl-terminal region const itutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a d omain that interacts with TFIIF74.