Logo Logo
Hilfe
Kontakt
Switch language to English
Structure and functional architecture of the Mediator middle module from budding yeast
Structure and functional architecture of the Mediator middle module from budding yeast
Mediator is a central coactivator complex required for regulated transcription by RNA polymerase (Pol) II in all eukaryotes. Budding yeast Mediator has a size of 1.4 MDa and consists of 25 subunits arranged in the head, middle, tail, and kinase modules. It is thought that Mediator forms an interface between the general RNA polymerase (RNA Pol) II machinery and transcriptional activators leading to promotion of pre-initiation complex (PIC) assembly. Mediator middle module from budding yeast consists of seven subunits Med1, 4, 7, 9, 10, 21, and 31 and was investigated during this thesis both structurally and functionally. Previously, the structure of a subcomplex comprising the C-terminal region of Med7 (Med7C) and Med21 was solved by X-ray crystallography and protocols for obtaining larger recombinant complexes were established in the laboratory. As structural and functional studies of Mediator are limited by the availability of protocols for the preparation of modules, I pursued these studies and established protocols for obtaining pure endogenous and recombinant complete Mediator middle module. Another subcomplex of the middle module, comprising the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1) was successfully crystallized and its structure solved during this work. It is found, that it contains a unique structure and acts also as a functional entity (termed submodule). The Med7N/31 submodule shows a novel fold, with two conserved proline-rich stretches in Med7N wrapping around the righthanded four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identified specific and overlapping functions of different Mediator submodules. Crystallization screening of larger middle module (sub-)complexes did not result in crystal formation, even after removal of some flexible regions. Thus alternative methods were applied to characterize the middle module topology. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion mobility mass spectrometry, limited proteolysis, light scattering, and small angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module, giving a potential explanation of why crystallization of larger complexes was unsuccessful. Moreover, based on systematic protein-protein interaction analysis, a new model for the subunit-subunit interaction network within the middle module of the Mediator is proposed. In this model, the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes Med7N/21, Med7C/31, and Med4/9. The subunits Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4. Furthermore, first steps in establishing an in vitro assay to test endogenous and recombinant middle module functionality have been initiated and will provide the basis for future studies.
Mediator of transcriptional regulation, RNA polymerase II transcription, gene regulation, coactivator, budding yeast, structure-function analysis, CTD, TFIIS
Koschubs, Tobias
2010
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Koschubs, Tobias (2010): Structure and functional architecture of the Mediator middle module from budding yeast. Dissertation, LMU München: Fakultät für Chemie und Pharmazie
[thumbnail of Koschubs_Tobias.pdf]
Vorschau
PDF
Koschubs_Tobias.pdf

19MB

Abstract

Mediator is a central coactivator complex required for regulated transcription by RNA polymerase (Pol) II in all eukaryotes. Budding yeast Mediator has a size of 1.4 MDa and consists of 25 subunits arranged in the head, middle, tail, and kinase modules. It is thought that Mediator forms an interface between the general RNA polymerase (RNA Pol) II machinery and transcriptional activators leading to promotion of pre-initiation complex (PIC) assembly. Mediator middle module from budding yeast consists of seven subunits Med1, 4, 7, 9, 10, 21, and 31 and was investigated during this thesis both structurally and functionally. Previously, the structure of a subcomplex comprising the C-terminal region of Med7 (Med7C) and Med21 was solved by X-ray crystallography and protocols for obtaining larger recombinant complexes were established in the laboratory. As structural and functional studies of Mediator are limited by the availability of protocols for the preparation of modules, I pursued these studies and established protocols for obtaining pure endogenous and recombinant complete Mediator middle module. Another subcomplex of the middle module, comprising the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1) was successfully crystallized and its structure solved during this work. It is found, that it contains a unique structure and acts also as a functional entity (termed submodule). The Med7N/31 submodule shows a novel fold, with two conserved proline-rich stretches in Med7N wrapping around the righthanded four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identified specific and overlapping functions of different Mediator submodules. Crystallization screening of larger middle module (sub-)complexes did not result in crystal formation, even after removal of some flexible regions. Thus alternative methods were applied to characterize the middle module topology. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion mobility mass spectrometry, limited proteolysis, light scattering, and small angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module, giving a potential explanation of why crystallization of larger complexes was unsuccessful. Moreover, based on systematic protein-protein interaction analysis, a new model for the subunit-subunit interaction network within the middle module of the Mediator is proposed. In this model, the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes Med7N/21, Med7C/31, and Med4/9. The subunits Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4. Furthermore, first steps in establishing an in vitro assay to test endogenous and recombinant middle module functionality have been initiated and will provide the basis for future studies.