Functional characterisation of a TATA binding protein (TBP) and a TBP interacting protein (TIP49a) in Giardia lamblia

The parasitic protozoan Giardia lamblia encodes a highly divergent TATA Binding Protein (gTBP) but Giardia gene promoters lack canonical TATA sequences. To investigate its DNA-binding activity, we expressed gTBP as a recombinant glutathione-S-transferase (GST) fusion protein. Electrophoretic mobility shift assays showed that the DNA-binding activity of GST-gTBP was determined by sequence-dependent structural features, rather than DNA sequence alone. Previous work in our laboratory identified the protein TIP49 among Giardia proteins that bind to a DNA element in core histone gene promoters. Since TIP49 in other organisms have helicase activity and is found in complexes with TBP, a recombinant GST-gTIP49a was prepared for its characterisation. We found that GST-gTIP49a bound preferentially to double stranded DNA in a sequence-independent manner, as did gTIP49a produced by thrombin protease removal of the GST tag. We also detected weak helicase activity of the gTIP49a in vitro. However, GST pulldown experiments with GST-gTBP and untagged gTIP49a did not show a direct interaction between these two proteins. Further research is required to precisely determine gTBP's DNA binding requirements, as well as to characterise gTIP49's activity and investigate the potential role of these two proteins in Giardia gene expression.;Keywords: Giardia lamblia, TATA Binding Protein (TBP), TBP Interacting Protein 49 (TIP49), DNA binding activity, helicase...