Date of Award
Doctor of Philosophy (PhD)
The process of transcription is investigated biochemically from several perspectives. First, a novel transcriptional activator, GaI4-IE, is shown to activate transcription in vitro from GaI4-DNA binding sites located upstream of two core promoters. The mechanism of activation is investigated by the development of a template competition assay. Using this assay, a partial preinitiation complex containing TFIIB is implicated as a target of GaI4-IE. These experiments, as well as others cited, suggest that activators affect early stages of PIC assembly, namely the formation of complexes including TFIID, TFIIA, and TFIIB. To further the study of this complex, attention is then turned to TFIIA. A eDNA is isolated encoding the small subunit of TFIIA from Drosophila melanogaster. The protein encoded by this eDNA, dTFIIA-S, is highly homologous to the cognate protein from yeast, as well as a protein encoded by a previously uncharacterized eDNA from rice, which likely encodes that organism's TFIIA-S. Using an antibody raised against recombinant dTFIIA-S, I show that dTFIIAS is present in Drosophila-derived nuclear extracts, and that the native dTFIIA-S migrates in SDS gels identically to recombinant dTFIIA-S, arguing against post-translational modification of the protein. In conjunction with a 55 kD precursor to the 35 and 19 kD subunits of human TFIIA, the functional properties of dTFIIA-S are investigated. These experiments show that dTFIIA-S is competent to form a bonafide DA promoter complex. Further, both the 55 kD precursor, and dTFIIA-S, are shown to be constituents of the DA promoter complex. Finally, it is shown that dTFIIA-S can reconstitute TFIIA transcripitonal activity in a reconstituted transcription system consisting of highly purified or recombinant factors including TFIID. Based on the above results, it is concluded that dTFIIA-S represents a functional TFIIA subunit, and that the combination of recombinant hTFIIA/ a and dTFIIA-S is sufficient to reconstitute functional TFIIA. The protein-protein interactions of the recombinant TFIIA subunits are then investigated. First, both subunits of TFIIA are shown to independently interact with both human and yeast TBP. In the case of yeast TBP, this interaction is impaired by a nonconservative double mutation in the TBP "basic region". This mutant TBP, which is correspondingly impaired in TBP-TFIIA promoter complex formation activity, is used to analyze the functional significance of the TBP-TFIIA interaction. These experiments show that the TBP-TFIIA interaction is required specifically in physiologic transcription systems containing TFIIA, but not in a TFIIA independent transcription system. Finally, interactions of TFIIA with preinitiation complex components and a novel cofactor are documented which suggest an intimate involvement of TFIIA in the process of transcriptional activation.
Bernstein, Richard Alan, "Preinitiation Complex Assembly and General Transcription Factor IIA" (1995). Student Theses and Dissertations. 330.