Getting insight into the structure of transcription factors and coactivators is an important step for understanding the mechanisms of gene expression at a molecular level. According to this we have been studying three transcription factors from the filamentous fungus Trichoderma reesei: (i) The TATA-box binding protein (TBP) a transcriptional factor which is essential for the formation of the preinitiation complex in general transcription machinery; (ii) The MBF1 protein (Multiprotein Bridging Factor 1)  known as a coactivator because it mediates the interaction of the TBP and gene-specifics regulators; (iii) The transcription factor BTF3 (RNA polymerase B Transcription Factor 3), which has many roles including the repression of a variety of genes, apoptosis, protein addressing and to be part of NAC (nascent polypeptide-associated complex) and complexes that interact with RNA polymerase II and DNA. The genomic structure of the TrMBF1, TrBTF3 and TrTBP genes was determined by analyzing the Trichoderma reesei genome and our EST database. These genes were cloned, over expressed in E. coli, and purified by using a chromatography strategy based on His-tag and Ni⁺²-chelating resins. Circular Dicroism (CD) spectra of TrBTF3 and TrMBF1 showed structural aspects that agreed with computational predictions. We are also performing a structural NMR analysis to solve the three dimensional structure of TrMBF1 and to study the structural details of its interaction with TrTBP.
Supporting: FAPESP.