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Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions./
Author:	Henley, Matthew.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
Description:	139 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-11, Section: B.
Contained By:	Dissertations Abstracts International81-11B.
Subject:	Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28006578
ISBN:	9798643184836

Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions.
Henley, Matthew.

Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 139 p.

Source: Dissertations Abstracts International, Volume: 81-11, Section: B.

Thesis (Ph.D.)--University of Michigan, 2020.

This item must not be sold to any third party vendors.

Transcription, the basal process by which all genes are regulated, is one of the most important tasks a cell undertakes. The molecular details of this process are well-studied, from the enzymatic action of RNA Polymerase II, the assembly of factors that load RNA Polymerase II onto a gene promoter, to the specific sets of transcriptional regulators that control the expression of individual genes. However, despite decades of study, the key regulatory interactions occurring between DNA-bound transcriptional activators and the transcriptional machinery are poorly understood. This is largely due to the preponderance of intrinsically disordered proteins involved in the process of transcriptional activation, leading to dynamic interactions difficult to characterize from a structural and mechanistic standpoint. Thus, a key challenge is to develop mechanistic models that explain how transcriptional activators are recognized by transcriptional machinery.Currently, molecular recognition models of transcriptional activators are based on analyses of a relatively small set of structurally related binding partners. The motivation behind this dissertation was therefore to dissect these recognition models by examining how a structurally divergent binding partner, namely the activator binding domain of Mediator subunit Med25, recognizes its activator binding partners.In chapter two, we dissect how Med25 forms binary and ternary complexes with a set of unrelated transcriptional activators. Using NMR and transient kinetic analysis, we find that Med25 uses conformational rearrangements along with two distinct binding interfaces to recognize partners with diverse sequences. Furthermore, kinetics experiments show that this mechanism of molecular recognition enables cooperative formation of ternary complexes with activators that bind to distinct sites. Molecular dynamics simulations demonstrate that, similar to the mechanisms of well-studied activator binding domains, conformational changes and allosteric communication are mediated by dynamic substructures in the activator binding domain. We establish the applicability of this observation to small molecule discovery by using disulfide-Tethering technology to discover a small molecule that covalently targets one of these dynamic substructures and induces allosteric effects that mimic natural activators.In chapter three, we examine the mechanisms by which Med25 forms complexes with a set of highly related activators. The common molecular recognition models of highly dynamic activator•coactivator interactions dictate that these "fuzzy" complexes are formed through entirely nonspecific mechanisms. In contrast, transient kinetics experiments demonstrate that small changes in the activator sequence result in redistribution of the activator•Med25 conformational ensemble, suggesting that the ensemble is controlled by specific intermolecular interactions. NMR analysis indicates the sensitivity of activator•Med25 conformational ensembles originates from specific interactions formed between the activator and the Med25 surface. Furthermore, this specific activator•Med25 recognition mechanism is enabled by the ability of the Med25 binding surface to remodel its conformation to complement the activator.This dissertation examines in detail how the structurally divergent activator binding domain of Med25 recognizes transcriptional activators and contributes to how activator molecular recognition is understood broadly. The work in this dissertation advances the conserved mechanistic role that coactivator conformational plasticity plays in molecular recognition and highlights a general framework for the development of small molecule modulators that exploit this mechanism. Finally, this work demonstrates that conformational remodeling of coactivators can play a key role even in the formation of exceptionally dynamic activator•coactivator complexes, suggesting that plasticity of folded binding partners is a critical element underlying molecular recognition of other dynamic protein-protein interactions.

ISBN: 9798643184836Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Transcription

Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions.
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245 1 0 $a Mechanistic Analysis of Disordered Transcriptional Protein-Protein Interactions.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2020
300 $a 139 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-11, Section: B.
500 $a Advisor: Mapp, Anna K.
502 $a Thesis (Ph.D.)--University of Michigan, 2020.
506 $a This item must not be sold to any third party vendors.
506 $a This item must not be added to any third party search indexes.
520 $a Transcription, the basal process by which all genes are regulated, is one of the most important tasks a cell undertakes. The molecular details of this process are well-studied, from the enzymatic action of RNA Polymerase II, the assembly of factors that load RNA Polymerase II onto a gene promoter, to the specific sets of transcriptional regulators that control the expression of individual genes. However, despite decades of study, the key regulatory interactions occurring between DNA-bound transcriptional activators and the transcriptional machinery are poorly understood. This is largely due to the preponderance of intrinsically disordered proteins involved in the process of transcriptional activation, leading to dynamic interactions difficult to characterize from a structural and mechanistic standpoint. Thus, a key challenge is to develop mechanistic models that explain how transcriptional activators are recognized by transcriptional machinery.Currently, molecular recognition models of transcriptional activators are based on analyses of a relatively small set of structurally related binding partners. The motivation behind this dissertation was therefore to dissect these recognition models by examining how a structurally divergent binding partner, namely the activator binding domain of Mediator subunit Med25, recognizes its activator binding partners.In chapter two, we dissect how Med25 forms binary and ternary complexes with a set of unrelated transcriptional activators. Using NMR and transient kinetic analysis, we find that Med25 uses conformational rearrangements along with two distinct binding interfaces to recognize partners with diverse sequences. Furthermore, kinetics experiments show that this mechanism of molecular recognition enables cooperative formation of ternary complexes with activators that bind to distinct sites. Molecular dynamics simulations demonstrate that, similar to the mechanisms of well-studied activator binding domains, conformational changes and allosteric communication are mediated by dynamic substructures in the activator binding domain. We establish the applicability of this observation to small molecule discovery by using disulfide-Tethering technology to discover a small molecule that covalently targets one of these dynamic substructures and induces allosteric effects that mimic natural activators.In chapter three, we examine the mechanisms by which Med25 forms complexes with a set of highly related activators. The common molecular recognition models of highly dynamic activator•coactivator interactions dictate that these "fuzzy" complexes are formed through entirely nonspecific mechanisms. In contrast, transient kinetics experiments demonstrate that small changes in the activator sequence result in redistribution of the activator•Med25 conformational ensemble, suggesting that the ensemble is controlled by specific intermolecular interactions. NMR analysis indicates the sensitivity of activator•Med25 conformational ensembles originates from specific interactions formed between the activator and the Med25 surface. Furthermore, this specific activator•Med25 recognition mechanism is enabled by the ability of the Med25 binding surface to remodel its conformation to complement the activator.This dissertation examines in detail how the structurally divergent activator binding domain of Med25 recognizes transcriptional activators and contributes to how activator molecular recognition is understood broadly. The work in this dissertation advances the conserved mechanistic role that coactivator conformational plasticity plays in molecular recognition and highlights a general framework for the development of small molecule modulators that exploit this mechanism. Finally, this work demonstrates that conformational remodeling of coactivators can play a key role even in the formation of exceptionally dynamic activator•coactivator complexes, suggesting that plasticity of folded binding partners is a critical element underlying molecular recognition of other dynamic protein-protein interactions.
590 $a School code: 0127.
650 4 $a Biochemistry. $3 518028
650 4 $a Biophysics. $3 518360
653 $a Transcription
653 $a Protein-protein interactions
653 $a Mechanistic Analysis
653 $a Fuzzy complexes
690 $a 0786
690 $a 0487
710 2 $a University of Michigan. $b Chemical Biology. $3 3546807
773 0 $t Dissertations Abstracts International $g 81-11B.
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791 $a Ph.D.
792 $a 2020
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28006578