1. Protein-protein interactions between widely-expressed and testis specific subunits of TFIIA and TFIID in Drosophila melanogaster Leah Hirschman, Mark Hiller Department of Biological Sciences Goucher College Abstract The regulation of transcription plays an important role in cellular development and differentiation. Spermatogenesis is an excellent model to explore the regulation of tissue-specific transcription. In Drosophila melanogaster, General Transcription Factors TFIIA and TFIID work together during transcription initiation. TFIID is a complex composed of TATA-binding protein (TBP) and fourteen TATA binding protein-associated factors (TAFs). TFIIA is comprised of three subunits, including the TFIIAγ subunit. In the testes, there is a homolog of the TFIIAγ subunit, which is encoded by the gene tfiia-s-2. Also in the testes, there are additional testis-TAFs (tTAFs) which are components of TFIID. Homologues of TBP, TBP-related factors 1 and 2 (TRF1 and TRF2), are found in the testes and other tissues. With so many different versions of proteins located in the testes, there are many possible combinations of proteins that could aid in the regulation of transcription in spermatogenesis. To study the possible interactions between widely expressed proteins and testis specific proteins, GST pull-down assays were used to analyze protein-protein interactions in vitro. By studying the interactions between proteins that regulate transcription in spermatogenesis, we will create a more comprehensive model of the complexes that regulate testis-specific transcription. Figure 1: Spermatogenesis is an excellent model system to explore tissue-specific transcription regulation. Spermatogenesis begins when a testis stem cell divides and one subsequent daughter cell undergoes four cycles of mitosis. After these mitotic divisions, high levels of transcription regulation occur. Following this growth and gene expression phase, cells undergo meiotic divisions and cellular differentiation to produce sperm cells. Figure 3: Model of the crystal structure derived from the TFIIA/TBP/DNA complex from yeast. TFIIA (red, blue, and green) and TATA-binding protein (TBP, a subunit of TFIID, orange) are known to interact with each other and with DNA (yellow) during transcription initiation. There is a possibility that testis-specific homologues of TFIIA (TFIIAγ-2), TBP homologues TRF1 and TRF2, and testis-specific TAF’s (tTAF’s) are also involved in the regulation of transcription during spermatogenesis. Figure 4: TRF1 and TRF2 proteins were synthesized in vitro using a transcription/translation system. Recombinant V5 epitope tagged TRF1 and TRF2 proteins were expressed in vitro and visualized by Immuno-blot using anti-V5 antibody. Lanes 1 and 2 are TRF1 while lanes 3 and 4 are TRF2. The high concentration lanes contain 3.75 times more protein than the low concentration lanes. One lane is used for the TRF V5-antibody control. A recombinant construct expressing TBP is under construction. Table 1: The General Transcription Factors TFIIA and TFIID are essential for transcription initiation. TFIID is comprised of TBP and TBP-associated factors (TAF’s). In many tissues, TBP homologues (TRF1 and TRF2) are expressed. Five TAF homologues are expressed only in the testis. TFIIA is comprised of three subunits: α, β, and γ. The testis-specific homologue TFIIAγ-2 is expressed in the testes and is encoded by the gene tfiia-s-2. Figure 5: GST pull down assays can detect protein- protein interactions between TBP/TAF homologues and TFIIA. E. coli expressed TFIIA is tagged with glutathione s-transferase (GST). GST tagged proteins will bind to glutathione-coated agarose beads and can be purified. Any proteins that interact with the GST tagged proteins will also remain bound to the beads and can be co-purified. Three forms of TFIIA are expressed in E. coli, including the widely expressed form and two testis-specific forms. GST pull down assays can detect interactions between more than two proteins. GST Pull Down 45.0 31.0 21.5 MW in kDa TRF1 TRF2 C L H L H TRF2 Figure 2: Amino acid alignment of TFIIA γ-proteins. The generally expressed Human (hTFIIAg) and fly (dTFIIA-S) TFIIA proteins are aligned along with testis-specific homologues. Two spliced forms (dTFIIA-S-2a and dTFIIA-S-2b) are shown. Black shading indicates identity while grey shading indicates similarity within the amino acid sequences. Sections α1 and α2 indicate α-helix secondary structure and sections β1-3 indicate β-sheet secondary structure. Testis- specific TFIIA-S-2b has extra amino acids in between sections α1 and α2 that are not seen in any other version of TFIIA. 1 2 3 4 5 Future Directions Continue to analyze protein-protein interactions in vitro using GST pull down assays Create a recombinant construct expressing TBP Create a more comprehensive model of the protein- protein complexes that aid in transcription regulation Acknowledgments: Goucher College for funding TRF1