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Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

Ellisdon AM, Dimitrova L, Hurt E, Stewart M - Nat. Struct. Mol. Biol. (2012)

Bottom Line: Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding.Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export.These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

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Structural basis for the Thp1–Sem1 interaction. (a,b) Interface between Thp1 and the C-terminal Sem1 binding region with (a) important Sem1 (yellow) sidechains shown as sticks and (b) hydrophobic Thp1 residues highlighted in green. (c,d) Corresponding views of the interface between the Sem1 N-terminal region (yellow) and Thp1 with (c) important sidechains shown as sticks and (d) the electrostatic surface potential of the N-terminal Sem1 binding site on Thp1. (e) PCID2 (residues 205-399, blue) and DSS1 (residues 38-67, orange) complex with the α-helices as cylinders and DSS1 in worm format. DSS1 sidechains that make key interactions within the interface are shown as sticks. (f) Structural alignment of the PCID2–DSS1 complex with the corresponding residues of Thp1 (grey) and Sem1 (yellow) from the Sac3–Thp1–Sem1 complex.
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Figure 2: Structural basis for the Thp1–Sem1 interaction. (a,b) Interface between Thp1 and the C-terminal Sem1 binding region with (a) important Sem1 (yellow) sidechains shown as sticks and (b) hydrophobic Thp1 residues highlighted in green. (c,d) Corresponding views of the interface between the Sem1 N-terminal region (yellow) and Thp1 with (c) important sidechains shown as sticks and (d) the electrostatic surface potential of the N-terminal Sem1 binding site on Thp1. (e) PCID2 (residues 205-399, blue) and DSS1 (residues 38-67, orange) complex with the α-helices as cylinders and DSS1 in worm format. DSS1 sidechains that make key interactions within the interface are shown as sticks. (f) Structural alignment of the PCID2–DSS1 complex with the corresponding residues of Thp1 (grey) and Sem1 (yellow) from the Sac3–Thp1–Sem1 complex.

Mentions: Sem1 makes extensive contacts with Thp1, burying 2741 Å2 of surface area, but makes only minor contact to Sac3, burying 293 Å2 (Supplementary Movie 2). Sem1 residues 53-89 make extensive interactions across the surface of the Thp1 superhelical domain and form a strongly conserved C-terminal helix (Supplementary Fig. 1) that binds in the cleft formed between helices α16 and α17 of the winged helix domain (Fig. 2a,b). The conserved Sem1 Trp60 and Trp64 are buried in surface pockets formed between residues on Thp1 helices α11, α12, and α16, whereas conserved hydrophobic residues (Phe73, Leu77, Leu81) lock the Sem1 helix into the hydrophobic cleft of the winged helix domain (Fig. 2b). The Sem1 helix and Trp60 have excellent density (Supplementary Fig. 2a) and low B-factors, suggesting that they provide the Sem1 binding register, whereas the negative charges of the aspartic and glutamic acids strengthen the interaction by forming ionic interactions and hydrogen bonds with the positively charged surface of Thp1. This intimate burial of the Sem1 C-terminus is consistent with C-terminally tagged Sem1 losing both its ability to bind Thp1 in yeast and its mRNA export function24. Sem1 residues 23-41 bind in a positively charged cleft formed by the TPR-like helices of the Thp1 superhelical domain (Fig. 2c,d). In this cleft, aspartates and glutamates from Sem1 form extensive ionic interactions and hydrogen bonds with several Thp1 helices (Fig 2c). Furthermore, the ring of conserved Sem1 Phe35 is buried. This region of Sem1 also makes limited contact with Sac3 helices α4 and α6, but these Sem1 residues are poorly conserved and probably contribute little to the overall binding. No electron density was observed for the poorly-conserved residues (1-22) at the Sem1 N-terminus, and between the N and C-terminal binding sites (residues 42-52).


Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

Ellisdon AM, Dimitrova L, Hurt E, Stewart M - Nat. Struct. Mol. Biol. (2012)

Structural basis for the Thp1–Sem1 interaction. (a,b) Interface between Thp1 and the C-terminal Sem1 binding region with (a) important Sem1 (yellow) sidechains shown as sticks and (b) hydrophobic Thp1 residues highlighted in green. (c,d) Corresponding views of the interface between the Sem1 N-terminal region (yellow) and Thp1 with (c) important sidechains shown as sticks and (d) the electrostatic surface potential of the N-terminal Sem1 binding site on Thp1. (e) PCID2 (residues 205-399, blue) and DSS1 (residues 38-67, orange) complex with the α-helices as cylinders and DSS1 in worm format. DSS1 sidechains that make key interactions within the interface are shown as sticks. (f) Structural alignment of the PCID2–DSS1 complex with the corresponding residues of Thp1 (grey) and Sem1 (yellow) from the Sac3–Thp1–Sem1 complex.
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Related In: Results  -  Collection

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Figure 2: Structural basis for the Thp1–Sem1 interaction. (a,b) Interface between Thp1 and the C-terminal Sem1 binding region with (a) important Sem1 (yellow) sidechains shown as sticks and (b) hydrophobic Thp1 residues highlighted in green. (c,d) Corresponding views of the interface between the Sem1 N-terminal region (yellow) and Thp1 with (c) important sidechains shown as sticks and (d) the electrostatic surface potential of the N-terminal Sem1 binding site on Thp1. (e) PCID2 (residues 205-399, blue) and DSS1 (residues 38-67, orange) complex with the α-helices as cylinders and DSS1 in worm format. DSS1 sidechains that make key interactions within the interface are shown as sticks. (f) Structural alignment of the PCID2–DSS1 complex with the corresponding residues of Thp1 (grey) and Sem1 (yellow) from the Sac3–Thp1–Sem1 complex.
Mentions: Sem1 makes extensive contacts with Thp1, burying 2741 Å2 of surface area, but makes only minor contact to Sac3, burying 293 Å2 (Supplementary Movie 2). Sem1 residues 53-89 make extensive interactions across the surface of the Thp1 superhelical domain and form a strongly conserved C-terminal helix (Supplementary Fig. 1) that binds in the cleft formed between helices α16 and α17 of the winged helix domain (Fig. 2a,b). The conserved Sem1 Trp60 and Trp64 are buried in surface pockets formed between residues on Thp1 helices α11, α12, and α16, whereas conserved hydrophobic residues (Phe73, Leu77, Leu81) lock the Sem1 helix into the hydrophobic cleft of the winged helix domain (Fig. 2b). The Sem1 helix and Trp60 have excellent density (Supplementary Fig. 2a) and low B-factors, suggesting that they provide the Sem1 binding register, whereas the negative charges of the aspartic and glutamic acids strengthen the interaction by forming ionic interactions and hydrogen bonds with the positively charged surface of Thp1. This intimate burial of the Sem1 C-terminus is consistent with C-terminally tagged Sem1 losing both its ability to bind Thp1 in yeast and its mRNA export function24. Sem1 residues 23-41 bind in a positively charged cleft formed by the TPR-like helices of the Thp1 superhelical domain (Fig. 2c,d). In this cleft, aspartates and glutamates from Sem1 form extensive ionic interactions and hydrogen bonds with several Thp1 helices (Fig 2c). Furthermore, the ring of conserved Sem1 Phe35 is buried. This region of Sem1 also makes limited contact with Sac3 helices α4 and α6, but these Sem1 residues are poorly conserved and probably contribute little to the overall binding. No electron density was observed for the poorly-conserved residues (1-22) at the Sem1 N-terminus, and between the N and C-terminal binding sites (residues 42-52).

Bottom Line: Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding.Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export.These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

ABSTRACT
The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

Show MeSH