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Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly.

Male G, von Appen A, Glatt S, Taylor NM, Cristovao M, Groetsch H, Beck M, Müller CW - Nat Commun (2015)

Bottom Line: How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood.We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138.The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Meyerhofstrasse 1, Heidelberg 69117, Germany.

ABSTRACT
In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

No MeSH data available.


Crystal structures of the TPR array of τ131 and the central extended winged helix (eWH) domain of τ138.(a) Schematic domain architecture of τ131. The TPR array is highlighted and coloured according to the solved crystal structure in b. Putative TPRs are indicated in grey. (b) Crystal structure of the τ131 (123–566) TPR array in ribbon representation. Two views are displayed, related by a 45° rotation. A dashed line indicates a region of the electron density where no residues could be built with confidence (residues 317–336). TPRs are numbered 1–10. (c) Schematic domain architecture of τ138. Predicted winged helix (WH) domains, the high mobility group (HMG)-box domain and the helical region are shaded in grey. The central eWH domain is shaded in dark green. The τIR is shaded in light green (see text for details). (d) Crystal structure of the τ138 (546–641) eWH domain in ribbon representation. Two views are displayed, related by a 180° rotation. A schematic of the arrangement of α-helices and β-strands is displayed underneath the structure.
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f2: Crystal structures of the TPR array of τ131 and the central extended winged helix (eWH) domain of τ138.(a) Schematic domain architecture of τ131. The TPR array is highlighted and coloured according to the solved crystal structure in b. Putative TPRs are indicated in grey. (b) Crystal structure of the τ131 (123–566) TPR array in ribbon representation. Two views are displayed, related by a 45° rotation. A dashed line indicates a region of the electron density where no residues could be built with confidence (residues 317–336). TPRs are numbered 1–10. (c) Schematic domain architecture of τ138. Predicted winged helix (WH) domains, the high mobility group (HMG)-box domain and the helical region are shaded in grey. The central eWH domain is shaded in dark green. The τIR is shaded in light green (see text for details). (d) Crystal structure of the τ138 (546–641) eWH domain in ribbon representation. Two views are displayed, related by a 180° rotation. A schematic of the arrangement of α-helices and β-strands is displayed underneath the structure.

Mentions: τ131 and τ138 are the largest subunits of the τA and τB subcomplexes, respectively5. τ131 was predicted to contain a highly conserved TPR array architecture at the N terminus with additional conserved TPRs predicted at the C terminus19 (Fig. 2a). Given that our crosslinking data implicated the TPR array of τ131 in connecting τA and τB, and the reported importance of this region for TFIIIB assembly, we determined the crystal structure of the TPR array (residues 123–566) in two different space groups (P62 and P43). Starting from a construct expressing residues 1–580 of τ131, we identified a stable fragment by limited proteolysis and mass spectrometry corresponding to residues 123–566. We expressed this truncated S. cerevisiae τ131 (123–566) protein in E. coli, purified it using affinity and size-exclusion chromatography and subsequently carried out crystallization trials. We were able to collect diffraction data up to 3.15 Å (P62) and 3.4 Å (P43) resolution. The lower-resolution structure was solved by multiple isomorphous replacement with anomalous signal (MIRAS), using anomalous signal from crystals prepared with selenomethionine substituted proteins or native crystals soaked with p-chloromercuribenzensulfonic acid to final Rwork/Rfree values of 20.9%/24.5% The higher-resolution structure was solved using selenomethionine substituted protein in single anomalous dispersion (SAD) experiments and subsequently refined to Rwork/Rfree values of 25.0%/28.7%. The combination of selenomethionine and mercury (Hg) markers aided the building of the structures. (Table 1, Supplementary Figs 2 and 3a and Supplementary Table 5).


Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly.

Male G, von Appen A, Glatt S, Taylor NM, Cristovao M, Groetsch H, Beck M, Müller CW - Nat Commun (2015)

Crystal structures of the TPR array of τ131 and the central extended winged helix (eWH) domain of τ138.(a) Schematic domain architecture of τ131. The TPR array is highlighted and coloured according to the solved crystal structure in b. Putative TPRs are indicated in grey. (b) Crystal structure of the τ131 (123–566) TPR array in ribbon representation. Two views are displayed, related by a 45° rotation. A dashed line indicates a region of the electron density where no residues could be built with confidence (residues 317–336). TPRs are numbered 1–10. (c) Schematic domain architecture of τ138. Predicted winged helix (WH) domains, the high mobility group (HMG)-box domain and the helical region are shaded in grey. The central eWH domain is shaded in dark green. The τIR is shaded in light green (see text for details). (d) Crystal structure of the τ138 (546–641) eWH domain in ribbon representation. Two views are displayed, related by a 180° rotation. A schematic of the arrangement of α-helices and β-strands is displayed underneath the structure.
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Related In: Results  -  Collection

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f2: Crystal structures of the TPR array of τ131 and the central extended winged helix (eWH) domain of τ138.(a) Schematic domain architecture of τ131. The TPR array is highlighted and coloured according to the solved crystal structure in b. Putative TPRs are indicated in grey. (b) Crystal structure of the τ131 (123–566) TPR array in ribbon representation. Two views are displayed, related by a 45° rotation. A dashed line indicates a region of the electron density where no residues could be built with confidence (residues 317–336). TPRs are numbered 1–10. (c) Schematic domain architecture of τ138. Predicted winged helix (WH) domains, the high mobility group (HMG)-box domain and the helical region are shaded in grey. The central eWH domain is shaded in dark green. The τIR is shaded in light green (see text for details). (d) Crystal structure of the τ138 (546–641) eWH domain in ribbon representation. Two views are displayed, related by a 180° rotation. A schematic of the arrangement of α-helices and β-strands is displayed underneath the structure.
Mentions: τ131 and τ138 are the largest subunits of the τA and τB subcomplexes, respectively5. τ131 was predicted to contain a highly conserved TPR array architecture at the N terminus with additional conserved TPRs predicted at the C terminus19 (Fig. 2a). Given that our crosslinking data implicated the TPR array of τ131 in connecting τA and τB, and the reported importance of this region for TFIIIB assembly, we determined the crystal structure of the TPR array (residues 123–566) in two different space groups (P62 and P43). Starting from a construct expressing residues 1–580 of τ131, we identified a stable fragment by limited proteolysis and mass spectrometry corresponding to residues 123–566. We expressed this truncated S. cerevisiae τ131 (123–566) protein in E. coli, purified it using affinity and size-exclusion chromatography and subsequently carried out crystallization trials. We were able to collect diffraction data up to 3.15 Å (P62) and 3.4 Å (P43) resolution. The lower-resolution structure was solved by multiple isomorphous replacement with anomalous signal (MIRAS), using anomalous signal from crystals prepared with selenomethionine substituted proteins or native crystals soaked with p-chloromercuribenzensulfonic acid to final Rwork/Rfree values of 20.9%/24.5% The higher-resolution structure was solved using selenomethionine substituted protein in single anomalous dispersion (SAD) experiments and subsequently refined to Rwork/Rfree values of 25.0%/28.7%. The combination of selenomethionine and mercury (Hg) markers aided the building of the structures. (Table 1, Supplementary Figs 2 and 3a and Supplementary Table 5).

Bottom Line: How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood.We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138.The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Meyerhofstrasse 1, Heidelberg 69117, Germany.

ABSTRACT
In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

No MeSH data available.