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Interaction of the mediator head module with RNA polymerase II.

ABSTRACT: Mediator, a large (21 polypeptides, MW ?1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.

SUBMITTER: Cai G

PROVIDER: S-EPMC3383055 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Interaction of the mediator head module with RNA polymerase II.

Cai Gang G Chaban Yuriy L YL Imasaki Tsuyoshi T Kovacs Julio A JA Calero Guillermo G Penczek Pawel A PA Takagi Yuichiro Y Asturias Francisco J FJ

Structure (London, England : 1993) 20120501 5

Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-a ...[more]

PMID: 22579255

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A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.

Project description:The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

| S-EPMC3648612 | biostudies-literature

The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

Project description:We use biochemically reconstituted transcription initiation components including the Mediator core and Cdk8 kinase modules (CKM) to investigate the function of the CKM in regulating the Mediator-RNA polymerase II interaction in a highly purified system. We use cross-linking coupled to mass spectrometry to map the interaction of the CKM and core Mediator, and in vitro phosphorylation assays followed by phosphopeptide enrichment coupled to mass spectrometry to identify Cdk8 phosphorylation targets in this context and biochemically dissect their functions. Finally, we investigate the function of phosphorylation by Cdk8 on transcription in vivo. Based on that, we propose a model to integrate the phosphorylation-dependent and -independent functions of the CKM in transcription initiation.

2021-04-28 | PXD023361 | Pride

The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II.

Project description:The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM-cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.

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Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II.

Project description:The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.

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The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

Project description:The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex Mediator that regulates RNA polymerase II (Pol II transcription. The CKM has negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. Here, we prepare recombinant CKM from the yeast Saccharomyces cerevisiae and show that it binds core Mediator (cMed) to sterically inhibit cMed binding to the Pol II preinitiation complex (PIC) in vitro. We further show that the Cdk8 kinase activity of CKM counteracts CKM-cMed interaction,thereby releasing CKM and enabling Mediator to bind the PIC. Finally, we report that the kinase activity of Cdk8 is required for gene activation during heat shock in vivo, but not under steady state growth conditions. These results converge with previous literature on a model for CKM function. In this model, CKM negatively regulates Mediator function at upstream activating sequences by preventing Mediator binding to the PIC at the promoter. During gene activation, Cdk8 kinase activity may release Mediator and allow its binding to the PIC, thereby stimulating transcription initiation and accounting for the positive function of CKM.

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RNA polymerase II - Mediator interaction

Project description:Comparison of genome-wide RNA polymerase II occupancy in wild-type and rpb3-2 yeast strains

2011-10-26 | E-MEXP-3043 | biostudies-arrayexpress

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II.

Project description:CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

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The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

Project description:The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

| PRJNA675650 | ENA

Activator-mediator binding stabilizes RNA polymerase II orientation within the human mediator-RNA polymerase II-TFIIF assembly.

Project description:The human Mediator complex controls RNA polymerase II (pol II) function in ways that remain incompletely understood. Activator-Mediator binding alters Mediator structure, and these activator-induced structural shifts appear to play key roles in regulating transcription. A recent cryo-electron microscopy (EM) analysis revealed that pol II adopted a stable orientation within a Mediator-pol II-TFIIF assembly in which Mediator was bound to the activation domain of viral protein 16 (VP16). Whereas TFIIF was shown to be important for orienting pol II within this assembly, the potential role of the activator was not assessed. To determine how activator binding might affect pol II orientation, we isolated human Mediator-pol II-TFIIF complexes in which Mediator was not bound to an activator. Cryo-EM analysis of this assembly, coupled with pol II crystal structure docking, revealed that pol II binds Mediator at the same general location; however, in contrast to VP16-bound Mediator, pol II does not appear to stably orient in the absence of an activator. Variability in pol II orientation might be important mechanistically, perhaps to enable sense and antisense transcription at human promoters. Because Mediator interacts extensively with pol II, these results suggest that Mediator structural shifts induced by activator binding help stably orient pol II prior to transcription initiation.

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Architecture of the Mediator head module.

Project description:Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

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