Structure and TBP binding of the Mediator head subcomplex Med8–Med18–Med20

The Mediator head module stimulates basal RNA polymerase II (Pol II) transcription and enables transcriptional regulation. Here we show that the head subunits Med8, Med18 and Med20 form a subcomplex (Med8/18/20) with two submodules. The highly conserved N-terminal domain of Med8 forms one submodule that binds the TATA box–binding protein (TBP) in vitro and is essential in vivo. The second submodule consists of the C-terminal region of Med8 (Med8C), Med18 and Med20. X-ray analysis of this submodule reveals that Med18 and Med20 form related β-barrel folds. A conserved putative protein-interaction face on the Med8C/18/20 submodule includes sites altered by srb mutations, which counteract defects resulting from Pol II truncation. Our results and published data support a positive role of the Med8/18/20 subcomplex in initiation-complex formation and suggest that the Mediator head contains a multipartite TBP-binding site that can be modulated by transcriptional activators.

[1]  M. Carey,et al.  Assembly of a Mediator/TFIID/TFIIA Complex Bypasses the Need for an Activator , 2003, Current Biology.

[2]  R. Young,et al.  Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. , 1989, Genetics.

[3]  M Ptashne,et al.  An activator target in the RNA polymerase II holoenzyme. , 1998, Molecular cell.

[4]  P. Cramer,et al.  Structure of the mediator subunit cyclin C and its implications for CDK8 function. , 2005, Journal of molecular biology.

[5]  David M. Chao,et al.  A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast , 1993, Cell.

[6]  R. Tjian,et al.  Transcriptional coactivator complexes. , 2001, Annual review of biochemistry.

[7]  Stewart Shuman,et al.  Structure and Mechanism of Yeast RNA Triphosphatase An Essential Component of the mRNA Capping Apparatus , 1999, Cell.

[8]  A. Danchin,et al.  Aeromonas hydrophila Adenylyl Cyclase 2: a New Class of Adenylyl Cyclases with Thermophilic Properties and Sequence Similarities to Proteins from Hyperthermophilic Archaebacteria , 1998, Journal of bacteriology.

[9]  R. Roeder,et al.  Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. , 2000, Trends in biochemical sciences.

[10]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[11]  Roger D. Kornberg,et al.  Mediator as a General Transcription Factor* , 2006, Journal of Biological Chemistry.

[12]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[13]  F. Holstege,et al.  A high resolution protein interaction map of the yeast Mediator complex. , 2004, Nucleic acids research.

[14]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[15]  Patrick Cramer,et al.  A Conserved Mediator Hinge Revealed in the Structure of the MED7·MED21 (Med7·Srb7) Heterodimer* , 2005, Journal of Biological Chemistry.

[16]  J. Ranish,et al.  Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. , 1999, Genes & development.

[17]  Shwu‐Yuan Wu,et al.  Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors , 2003, Molecular and Cellular Biology.

[18]  E. Geiduschek,et al.  Crystal structure of a transcription factor IIIB core interface ternary complex , 2003, Nature.

[19]  C. Eckerskorn,et al.  High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. , 1995, European journal of biochemistry.

[20]  M. Boube,et al.  Evidence for a Mediator of RNA Polymerase II Transcriptional Regulation Conserved from Yeast to Man , 2002, Cell.

[21]  P. Cramer,et al.  An Extended Winged Helix Domain in General Transcription Factor E/IIEα* , 2003, Journal of Biological Chemistry.

[22]  C. Gustafsson,et al.  The mediator complex. , 2004, Advances in protein chemistry.

[23]  Roger D Kornberg,et al.  Mediator and the mechanism of transcriptional activation. , 2005, Trends in biochemical sciences.

[24]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[25]  Kevin Struhl,et al.  A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. , 2004, Molecular cell.

[26]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[27]  P. Lijnzaad,et al.  Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA. , 2006, Molecular cell.

[28]  Sang Jun Han,et al.  The Structural and Functional Organization of the Yeast Mediator Complex* , 2001, The Journal of Biological Chemistry.

[29]  C. Sander,et al.  Dali: a network tool for protein structure comparison. , 1995, Trends in biochemical sciences.

[30]  S. Han,et al.  An Activator Binding Module of Yeast RNA Polymerase II Holoenzyme , 1999, Molecular and Cellular Biology.

[31]  L. Aravind,et al.  The catalytic domains of thiamine triphosphatase and CyaB-like adenylyl cyclase define a novel superfamily of domains that bind organic phosphates , 2002, BMC Genomics.

[32]  R. Kornberg,et al.  Structural organization of yeast and mammalian mediator complexes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Translation Functions Biological Crystallography Likelihood-enhanced Fast Translation Functions , 2022 .

[34]  A. Berk,et al.  Activation domain–mediator interactions promote transcription preinitiation complex assembly on promoter DNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Sohail Malik,et al.  Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Steven Hahn,et al.  A transcription reinitiation intermediate that is stabilized by activator , 2000, Nature.

[37]  John J. Wyrick,et al.  Interplay of Positive and Negative Regulators in Transcription Initiation by RNA Polymerase II Holoenzyme , 1998, Molecular and Cellular Biology.

[38]  Patrice Gouet,et al.  ESPript: analysis of multiple sequence alignments in PostScript , 1999, Bioinform..

[39]  C. Gustafsson,et al.  Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions. , 2006, Molecular cell.

[40]  Y. Liu,et al.  Yeast Nuclear Extract Contains Two Major Forms of RNA Polymerase II Mediator Complexes* , 2001, The Journal of Biological Chemistry.

[41]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[42]  N. Oppenheimer,et al.  Structure and mechanism , 1989 .

[43]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[44]  P. Cramer,et al.  Architecture of initiation-competent 12-subunit RNA polymerase II , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Robert Tjian,et al.  Structure, Function, and Activator-Induced Conformations of the CRSP Coactivator , 2002, Science.

[46]  Thessa T. J. P. Kockelkorn,et al.  Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. , 2005, Molecular cell.

[47]  R. Young,et al.  A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID , 1992, Cell.