Structure and in vivo requirement of the yeast Spt6 SH2 domain.

During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.

[1]  F. Winston,et al.  Evidence That Spt6p Controls Chromatin Structure by a Direct Interaction with Histones , 1996, Science.

[2]  F. Winston,et al.  SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. , 1992, Genetics.

[3]  F. Winston,et al.  SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus , 1990, Molecular and cellular biology.

[4]  R. Evans,et al.  The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. , 2007, Genes & development.

[5]  Peer Bork,et al.  SMART 5: domains in the context of genomes and networks , 2005, Nucleic Acids Res..

[6]  W. Webb,et al.  Spt6 enhances the elongation rate of RNA polymerase II in vivo , 2009, The EMBO journal.

[7]  B. Strahl,et al.  Roles for Ctk1 and Spt6 in Regulating the Different Methylation States of Histone H3 Lysine 36 , 2008, Molecular and Cellular Biology.

[8]  C. Müller,et al.  Structure of an activated Dictyostelium STAT in its DNA-unbound form. , 2004, Molecular cell.

[9]  T. Pawson,et al.  The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. , 2006, Molecular cell.

[10]  O. Witte,et al.  BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner , 1991, Cell.

[11]  D. Baltimore,et al.  Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides , 1993, Nature.

[12]  J. Lis,et al.  High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. , 2000, Genes & development.

[13]  Patrick J. Killion,et al.  Genetic reconstruction of a functional transcriptional regulatory network , 2007, Nature Genetics.

[14]  T Pawson,et al.  SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. , 1991, Science.

[15]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[16]  T Pawson,et al.  SH2 domains, interaction modules and cellular wiring. , 2001, Trends in cell biology.

[17]  F. Winston,et al.  Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. , 1998, Genes & development.

[18]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[19]  M. P. Cummings.  PHYLIP (Phylogeny Inference Package) , 2004 .

[20]  Xin-Yuan Fu,et al.  Identification of the Linker-SH2 Domain of STAT as the Origin of the SH2 Domain Using Two-dimensional Structural Alignment* , 2004, Molecular & Cellular Proteomics.

[21]  G. Fink,et al.  Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. , 1984, Genetics.

[22]  Rodrigo Lopez,et al.  Web Services at the European Bioinformatics Institute , 2007, Nucleic Acids Res..

[23]  G. Shaw,et al.  A yeast SH2 domain. , 1993, Trends in biochemical sciences.

[24]  D. Reinberg,et al.  Human Spt6 Stimulates Transcription Elongation by RNA Polymerase II In Vitro , 2004, Molecular and Cellular Biology.

[25]  L. Eichinger,et al.  Manifestations of multicellularity: Dictyostelium reports in. , 2005, Trends in genetics : TIG.

[26]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[27]  Gavin Sherlock,et al.  The Longhorn Array Database (LAD): An Open-Source, MIAME compliant implementation of the Stanford Microarray Database (SMD) , 2003, BMC Bioinformatics.

[28]  J. Tyler,et al.  Transcriptional activators are dispensable for transcription in the absence of Spt6-mediated chromatin reassembly of promoter regions. , 2006, Molecular cell.

[29]  R. Aebersold,et al.  The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes , 2007, Proceedings of the National Academy of Sciences.

[30]  Ronald W. Davis,et al.  A high-resolution atlas of nucleosome occupancy in yeast , 2007, Nature Genetics.

[31]  D Cowburn,et al.  Modular peptide recognition domains in eukaryotic signaling. , 1997, Annual review of biophysics and biomolecular structure.

[32]  David L. Steffen,et al.  The genome of the social amoeba Dictyostelium discoideum , 2005, Nature.

[33]  S. Hubbard,et al.  Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor. , 2003, Molecular cell.

[34]  T. Hughes,et al.  Chromatin- and Transcription-Related Factors Repress Transcription from within Coding Regions throughout the Saccharomyces cerevisiae Genome , 2008, PLoS biology.

[35]  Michael Knop,et al.  A versatile toolbox for PCR‐based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes , 2004, Yeast.

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

[37]  Qi Zheng,et al.  GOEAST: a web-based software toolkit for Gene Ontology enrichment analysis , 2008, Nucleic Acids Res..

[38]  J. Jaehning,et al.  Ctr9, Rtf1, and Leo1 Are Components of the Paf1/RNA Polymerase II Complex , 2002, Molecular and Cellular Biology.

[39]  K. Jones,et al.  The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. , 2008, Genes & development.

[40]  Bernd Groner,et al.  TRANSCRIPTION FACTOR STAT3B/DNA COMPLEX , 1999 .

[41]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[42]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[43]  J. Darnell,et al.  Crystal Structure of a Tyrosine Phosphorylated STAT-1 Dimer Bound to DNA , 1998, Cell.

[44]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.

[45]  J. R. Morris,et al.  Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. , 2000, Genes & development.

[46]  Thomas R. Schneider,et al.  HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs , 2004 .

[47]  Craig D. Kaplan,et al.  Transcription Elongation Factors Repress Transcription Initiation from Cryptic Sites , 2003, Science.

[48]  F. Winston,et al.  The SPT6 gene is essential for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae , 1987, Molecular and cellular biology.

[49]  S. Becker,et al.  Three-dimensional structure of the Stat3β homodimer bound to DNA , 1998, Nature.

[50]  K. Arndt,et al.  Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. , 2000, Genetics.

[51]  T. Fuchs,et al.  A new gene locus of Bordetella pertussis defines a novel family of prokaryotic transcriptional accessory proteins , 1996, Journal of bacteriology.

[52]  D. Cowburn,et al.  Binding of a High Affinity Phosphotyrosyl Peptide to the Src SH 2 Domain : Crystal Structures of the Complexed and Peptide , 2004 .

[53]  Craig D. Kaplan,et al.  Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus* , 2005, Journal of Biological Chemistry.

[54]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[55]  Benjamin Guglielmi,et al.  TFIIS elongation factor and Mediator act in conjunction during transcription initiation in vivo , 2007, Proceedings of the National Academy of Sciences.

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

[57]  A. Ducruix,et al.  Crystal structure of the mammalian Grb2 adaptor. , 1995, Science.

[58]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[59]  Airlie J. McCoy,et al.  Solving structures of protein complexes by molecular replacement with Phaser , 2006, Acta crystallographica. Section D, Biological crystallography.

[60]  T. Pawson,et al.  A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction , 1992, Molecular and cellular biology.

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

[62]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[63]  P. Cramer,et al.  Identification, structure, and functional requirement of the Mediator submodule Med7N/31 , 2009, The EMBO journal.

[64]  Sean J. Johnson,et al.  Crystal Structure and RNA Binding of the Tex Protein from Pseudomonas aeruginosa , 2008, Journal of molecular biology.

[65]  Thomas C Terwilliger,et al.  Automated structure solution, density modification and model building. , 2002, Acta crystallographica. Section D, Biological crystallography.

[66]  A I Saeed,et al.  TM4: a free, open-source system for microarray data management and analysis. , 2003, BioTechniques.

[67]  Maria Jesus Martin,et al.  The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003 , 2003, Nucleic Acids Res..

[68]  G. Cagney,et al.  RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach , 2002, Molecular and Cellular Biology.

[69]  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.

[70]  T Pawson,et al.  Structural basis for specificity switching of the Src SH2 domain. , 2000, Molecular cell.

[71]  Chuong B. Do,et al.  ProbCons: Probabilistic consistency-based multiple sequence alignment. , 2005, Genome research.

[72]  S. Harrison,et al.  Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes , 1996, Nature Structural Biology.