UBC Theses and Dissertations

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UBC Theses and Dissertations

Biophysical insights into the regulatory and DNA-binding mechanisms of the eukaryotic transcription factors Pax5 and Ets1 Perez-Borrajero, Cecilia

Abstract

Transcription factors are proteins that bind at promoter and enhancer sites to regulate gene expression. In this thesis, I used NMR spectroscopy and other methods to investigate the structural and biophysical basis of DNA binding by two eukaryotic transcription factors that are crucial in the development of lymphocytes, Pax5 and Ets1. In chapter 2, I describe how the two subdomains comprising the bipartite DNA-binding Paired domain of Pax5 cooperate to mediate transcriptional regulation. The N-terminal subdomain recognizes DNA sequences in a highly specific manner, whereas the C-terminal subdomain shows little sequence discrimination. The more rigid C-terminal subdomain binds DNA primarily though non-specific electrostatic interactions. In contrast, association with specific DNAs by the dynamic N-terminal subdomain involves relatively large and compensating changes in enthalpy and entropy that point to structural rearrangements upon binding. I propose that the distinct behaviors of the subdomains allow the Pax5 protein to rapidly scan non-specific genomic DNA while retaining specificity for functional regulatory sites. In chapter 3, I expand our understanding of the structural and thermodynamic basis of Ets1 autoinhibition. Previously it was reported that an intrinsically disordered serine-rich region (SRR) interacts transiently with the adjacent ETS domain to attenuate DNA binding. Although forming a dynamic fuzzy complex, I was able to use NMR spectroscopy and X-ray crystallography to provide a detailed mechanism for this inhibitory interaction. In particular, I exploited a trans peptide system to show that the SRR uses a combination of electrostatic and hydrophobic-driven interactions to sterically block the ETS domain DNA-binding interface. I also show how phosphorylation of the SRR strengthens its association with the ETS domain. Altogether, these results explain how the activity of Ets1 is regulated at the level of DNA binding through post-translational modifications that impinge upon the SRR.

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