Structural characterization of DNA binding and autoinhibition by the Ets1 transcription factor
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Desjardins, Geneviève
issued: 2015-04-01T16:27:25Z
issued: 2015
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: Ets1 belongs to the ETS transcription factor family and plays key roles in regulating eukaryotic gene expression. The affinity of the Ets1 for its cognate DNA sites is autoinhibited by an intrinsically disordered serine-rich region (SRR) and an appended helical inhibitory module (IM). Through transient interactions, the SRR both sterically blocks the ETS domain and allosterically stabilizes the IM to modulate DNA-binding affinity. Calmodulin-dependent kinase II phosphorylation of five serines within the SRR progressively reinforces autoinhibition in response to calcium signaling. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine/tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively-charged DNA recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression. We also investigated by NMR spectroscopy the interaction of Ets1 with specific and nonspecific oligonucleotides. Upon binding DNA, helices HI-1 and HI-2 of the IM unfold. Thus, autoinibition does not impart DNA-binding specificity. Using amide chemical shift perturbation mapping, we also show that Ets1 binds both specific and non-specific olig…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/52606
note: STRUCTURAL CHARACTERIZATION OF DNA BINDING AND AUTOINHIBITION BY THE ETS1 TRANSCRIPTION FACTOR by Geneviève Desjardins BA, Université de Montréal, 2005 Ms. Sc., Université de Montréal, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2015 Geneviève Desjardins, 2015 ii Abstract Ets1 belongs to the ETS transcription factor family and plays key roles in regulating eukaryotic gene expression. The affinity of the Ets1 for its cognate DNA sites is autoinhibited by an intrinsically disordered serine-rich region (SRR) and an appended helical inhibitory module (IM). Through transient interactions, the SRR both sterically blocks the ETS domain and allosterically stabilizes the IM to modulate DNA-binding affinity. Calmodulin-dependent kinase II phosphorylation of five serines within the SRR progressively reinforces autoinhibition in response to calcium signaling. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine/tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively-charged DNA recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their sy…
has type: Thesis/Dissertation
date issued: 2015-05
is shown at: 10.14288/1.0166107
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
rights URI: http://creativecommons.org/licenses/by-nc-nd/2.5/ca/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/52606