title: The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region.
abstract: Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Sem1 bind to the Sac3(M) region (residues ∼100-551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3(CID) region (residues ∼710-805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3(M) region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3(CID):Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100kDa, a 5.3Å resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative α-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9Å resolution structure obtained by X-ray crystallography.
  
  
  
  We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation.
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Aibara
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Xiao-Chen
authors.initials: XC
authors.lastname: Bai
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom. Electronic address: ms@mrc-lmb.cam.ac.uk.
authors.forename: Murray
authors.initials: M
authors.lastname: Stewart
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D008958:Models, Molecular; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry*; D018272:Porins / Q000737:chemistry*; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D053838:Scattering, Small Angle; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D029543:Nucleocytoplasmic Transport Proteins; D018272:Porins; C103382:SAC3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005090:Exodeoxyribonucleases
keywords: Cryo-EM; Crystallography; TREX-2 complex; mRNA nuclear export