The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region

Shintaro Aibara, Xiao Chen Bai, Murray Stewart

Research output: Contribution to journalArticle

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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 Sac3M region (residues ∼100–551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3CID 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 Sac3M region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3CID:Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100 kDa, a 5.3 Å 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 4.9 Å resolution structure obtained by X-ray crystallography. Summary statement 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.

Original languageEnglish (US)
Pages (from-to)316-324
Number of pages9
JournalJournal of Structural Biology
Volume195
Issue number3
DOIs
StatePublished - Sep 1 2016

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Saccharomyces cerevisiae
X Ray Crystallography
Sf9 Cells
Cell Nucleus Active Transport
Baculoviridae
Electron Microscopy
X-Rays
Messenger RNA
Genes
messenger ribonucleoprotein

Keywords

  • Cryo-EM
  • Crystallography
  • mRNA nuclear export
  • TREX-2 complex

ASJC Scopus subject areas

  • Structural Biology

Cite this

The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region. / Aibara, Shintaro; Bai, Xiao Chen; Stewart, Murray.

In: Journal of Structural Biology, Vol. 195, No. 3, 01.09.2016, p. 316-324.

Research output: Contribution to journalArticle

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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 Sac3M region (residues ∼100–551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3CID 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 Sac3M region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3CID:Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100 kDa, a 5.3 {\AA} 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 4.9 {\AA} resolution structure obtained by X-ray crystallography. Summary statement 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.",
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AB - 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 Sac3M region (residues ∼100–551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3CID 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 Sac3M region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3CID:Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100 kDa, a 5.3 Å 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 4.9 Å resolution structure obtained by X-ray crystallography. Summary statement 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.

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