Regulation of Ets-1 DNA binding

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Title Regulation of Ets-1 DNA binding
Publication Type dissertation
School or College School of Medicine
Department Oncological Sciences
Author Pufall, Miles August
Date 2004-12
Description Autoinhibitory domains are increasingly being recognized as important means for proteins to regulate their activity in cis based on cellular cues. The transcription factor Ets-1 is regulated by an autoinhibitory module that represses the DNA binding of the ETS domain by hindering the structural rearrangement that accompanies DNA binding. However, the autoinhibitory module does not simply inhibiting binding; it serves as a versatile integrator of cell signals, including Ca2+-induced phosphorylation. This thesis explores the mechanism of autoinhibition of DNA binding in Ets-1, particularly in response to phosphorylation. Initial structural studies define the interface between the autoinhibitory module and the ETS domain. A purified multiply phosphorylated Ets-1 fragment, ?N2445P, is used to demonstrate that phosphorylation induced stabilization of the ETS domain and inhibitory module are correlated with a reduction in DNA-binding affinity. NMR spectrometry experiments reveal a dynamic hydrophobic network that connects the inhibitory module to the DNA-binding interface to form a concerted, regulatable unit. Together, these data support an allosteric model of Ets-1 DNA binding in which a dynamic hydrophobic network translates the level of phosphorylation into DNA-binding affinity by modulating the equilibrium between active and inactive conformations. Mutational analysis of the phosphorylated serine rich region indicates that multiple phosphates contribute additively to the inhibition, allowing variable control of DNA binding depending on phosphorylation state. Surprisingly, the serine rich region, which serves as an allosteric effector, is unstructured and highly mobile. Thus, rather than forming a simple on/off switch, this allosteric mechanism allows adjustable control of activity by combining flexible and folded modules. These results suggest that conformation coupling to transient interactions may provide a general mechanism of action of flexible regulatory segments.
Type Text
Publisher University of Utah
Subject MESH Transcription, Genetic; Protein Binding
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Regulation of Ets-1 DNA binding". Spencer S. Eccles Health Sciences Library.
Rights Management © Miles August Pufall.
Format application/pdf
Format Medium application/pdf
Format Extent 5,157,233 bytes
Identifier undthes,4184
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available)
Master File Extent 5,157,273 bytes
ARK ark:/87278/s6cj8g6z
Setname ir_etd
ID 190394
Reference URL https://collections.lib.utah.edu/ark:/87278/s6cj8g6z
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