Article thumbnail

Preparation and topology of the Mediator middle module

By Tobias Koschubs, Kristina Lorenzen, Sonja Baumli, Saana Sandström, Albert J. R. Heck and Patrick Cramer

Abstract

Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. 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. Protein–protein interaction assays combined with previously published data suggest that 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

Topics: Gene Regulation, Chromatin and Epigenetics
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:2879511
Provided by: PubMed Central

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.

Suggested articles

Citations

  1. (2006). A Consensus Data Mining secondary structure prediction by combining GOR V and Fragment Database Mining.
  2. (2005). A conserved mediator hinge revealed in the structure of the MED7.MED21
  3. (2004). A high resolution protein interaction map of the yeast Mediator complex.
  4. (1991). A mediator required for activation of RNA polymerase II transcription in vitro.
  5. (1990). A novel mediator between activator proteins and the RNA polymerase II transcription apparatus.
  6. (2008). A nuclear receptor-like pathway regulating multidrug resistance in fungi.
  7. (2004). A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.
  8. (2006). An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis.
  9. (2007). An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument.
  10. (2006). ATSAS 2.1, a program package for small-angle scattering data analysis.
  11. (2008). Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex.
  12. (1999). Conserved structures of mediator and RNA polymerase II holoenzyme.
  13. (2004). Distinct conformational states of nuclear receptor-bound CRSP-Med complexes.
  14. (2002). Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man.
  15. (2006). Head module control of mediator interactions.
  16. (2008). I-TASSER server for protein 3D structure prediction.
  17. (2009). Identification, structure, and functional requirement of the Mediator submodule Med7N/31.
  18. (2010). Ion mobility mass spectrometry of proteins and protein assemblies.
  19. (2008). Ion mobility-mass spectrometry analysis of large protein complexes.
  20. (2008). Malleable machines in transcription regulation: the mediator complex.
  21. (2006). Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes.
  22. (2007). Med19(Rox3) regulates intermodule interactions in the Saccharomyces cerevisiae mediator complex.
  23. (2005). Mediator and the mechanism of transcriptional activation.
  24. (2005). Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets.
  25. (2009). Mediator structural conservation and implications for the regulation mechanism.
  26. (2008). Native mass spectrometry: a bridge between interactomics and structural biology.
  27. (1999). Protein secondary structure prediction based on position-specific scoring matrices.
  28. (2007). Purification of a plant Mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit.
  29. (2009). Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation.
  30. (2008). Stability and shape of hepatitis B virus capsids in vacuo.
  31. (2007). Structural biology of RNA polymerase III: mass spectrometry elucidates subcomplex architecture.
  32. (2005). Structure of the mediator subunit cyclin C and its implications for CDK8 function.
  33. (2002). Structure of the yeast RNA polymerase II holoenzyme: mediator conformation and polymerase interaction.
  34. (2008). Structure-system correlation identifies a gene regulatory Mediator submodule.
  35. (2006). The classical srb4-138 mutant allele causes dissociation of yeast Mediator.
  36. (2006). The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II.
  37. (2005). The HHpred interactive server for protein homology detection and structure prediction.
  38. (2009). The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function.
  39. (2007). The role of mass spectrometry in structure elucidation of dynamic protein complexes.
  40. (2001). The structural and functional organization of the yeast mediator complex.
  41. (2005). The structural and functional role of Med5 in the yeast Mediator tail module.
  42. (2005). The yeast Mediator complex and its regulation.
  43. (2001). Transcriptional coactivator complexes.
  44. (2000). Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells.
  45. (2003). Uniqueness of ab initio shape determination in small-angle scattering.
  46. (2001). Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes.