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 arm of the third chromosome. The protein encoded by the cDNA, termed dTFIIF30, was synthesized in Escherichia coli and purified to greater than 95% homogeneity. In reconstituted transcription reactions with purified basal factors, the specific activity of dTFIIF30 was identical to that of its human homologue. Moreover, a carboxyl-terminal fragment, designated dF30(119-276), which contains the carboxyl-terminal 158 amino 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 TFIIF74, 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. These results, combined with those of previous work on TFIIF, support a model for TFIIF30 function in which the carboxylterminal region constitutes a functional domain that can interact with RNA polymerase II to mediate basal transcription, whereas the amino terminus comprises a domain that interacts with TFIIF74.
Frank
D J
DJ
Department of Biology, University of California at San Diego, La Jolla 92093-0347.
Tyree
C M
CM
George
C P
CP
Kadonaga
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D014981:Xenopus
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF