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UBC Theses and Dissertations

Structural studies of protein partnerships of the PNT domain of GABP[alpha] MacIntosh, Scott Earl

Abstract

Background: The Ets family of transcription factors is an established model system for studying the regulation of gene expression. In addition to the DNA-binding ETS domain, several Ets proteins contain a second region of conserved sequence, the Pointed (PNT) domain. Although the exact functions of this domain remain to be established, there is a mounting body of evidence that the PNT domain mediates interactions with partner transcription factors. However, a detailed structural study of PNT domain interactions has yet to be carried out. This information is essential for a complete understanding of the role of the PNT domain in the control of gene expression by the Ets transcription factors. Goals: Previous work in the McIntosh laboratory has focused on the PNT domain of GABPα. To continue studies on this domain, three main goals of this thesis work were formulated: i) Use of a yeast two-hybrid system to screen for protein, partners of the domain. ii) A detailed study of the domain to investigate the possibility of self-association. iii) A detailed study of the domain to investigate the possibility of a small molecule binding to the domain. Results and conclusions: A yeast two-hybrid screen was used in an attempt to identify proteins that interacted with the minimal PNT domain of GABPα. One potential interacting protein, PBP, was identified. However, attempts to demonstrate a direct interaction between the proteins in vitro were unsuccessful. The behaviour of a minimal GABPα PNT domain construct during purification suggested that this protein formed oligomers. However, no evidence of self-association of the protein was obtained using several different techniques. As a result, this is likely an artifact with no biological relevance. Resonances not assignable to any amino acid residues were present in the NMR spectra of the GABPα PNT domain, raising the possibility that a small molecule was binding to the domain. Using NMR and mass spectrometry, this molecule was tentatively identified as an N-acetyl hexosamine oligomer. However, NMR relaxation experiments and further purification of the protein failed to provide any evidence that this molecule was binding the PNT domain of GABPα with appreciable affinity.

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