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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.


Related in: MedlinePlus

A binding hotspot on the τ131 TPR array for τ138 and Bdp1.(a) Mapped mutants of the τ131 TPR array (see text for details). (b) Sequence alignment of TPR 8. Identical residues are boxed in brick red, highly conserved in purple, medium conserved in pink and low conserved in white. Coloured arrowheads indicate the five mutated residues. (c) Close-up from the structure of TPR 8. The five residues selected for mutation are displayed as sticks; carbon atoms (grey); oxygen atoms (red). (d) Summary of ITC measurements using indicated τ131 (123–566) point mutants with τ138 (τIR) or τ138 (eWH-τIR). Wild-type (wt) measurements are included for reference. (e) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged τ138 (eWH-τIR). (−) indicates a background control for nonspecific binding of τ138 to the GST-affinity resin. A mixture of purified GST and untagged τ138 was also used as a negative control. Lower gel shows 5% of the input and upper gel shows bound fractions. (f) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged Bdp1. Negative controls and gel format as in e.
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f4: A binding hotspot on the τ131 TPR array for τ138 and Bdp1.(a) Mapped mutants of the τ131 TPR array (see text for details). (b) Sequence alignment of TPR 8. Identical residues are boxed in brick red, highly conserved in purple, medium conserved in pink and low conserved in white. Coloured arrowheads indicate the five mutated residues. (c) Close-up from the structure of TPR 8. The five residues selected for mutation are displayed as sticks; carbon atoms (grey); oxygen atoms (red). (d) Summary of ITC measurements using indicated τ131 (123–566) point mutants with τ138 (τIR) or τ138 (eWH-τIR). Wild-type (wt) measurements are included for reference. (e) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged τ138 (eWH-τIR). (−) indicates a background control for nonspecific binding of τ138 to the GST-affinity resin. A mixture of purified GST and untagged τ138 was also used as a negative control. Lower gel shows 5% of the input and upper gel shows bound fractions. (f) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged Bdp1. Negative controls and gel format as in e.

Mentions: Our structure of the τ131 TPR array allows us, for the first time, to map and analyse mutations that have been previously described (Fig. 4a). In detail, mutations that increase Pol III transcription cluster mostly on TPR 2 (ref. 26), those that decrease Pol III transcription spread over TPRs 8–10 (ref. 22) and those that rescue a τ138 temperature-sensitive mutation, map mostly to TPRs 7–8 (ref. 16). Having mapped the critical τ131 interaction region of τ138 to ∼50 amino acids, we next questioned where the τIR binds on the TPR array. Despite extensive efforts, we were unable to obtain structural information of the τ131–τIR complex although both polypeptides form a stable complex during size-exclusion chromatography. Instead, we analysed previously described mutations together with the surface conservation of the TPR array (Supplementary Fig. 3e), to predict where the τIR may bind. We expressed and purified five τ131 point mutants that all cluster on TPR 8 (Fig. 4b). With the exception of residue L469, all of these residues are acidic and surface exposed (Fig. 4c). The purified mutants all eluted at the same volume from a size-exclusion column when compared with the wild type, indicating that the mutations did not cause a destabilization of the proteins (Supplementary Fig. 7a). Using ITC, we determined binding affinities of the mutant τ131 TPR arrays to the τIR and the eWH-τIR (Fig. 4d and Supplementary Fig. 7b). No binding of the mutants D468K and L469K to the τ138 proteins could be detected by ITC. Mutants E472K and E498K showed much weaker binding, while the E497K mutant showed no significant decrease in binding affinity to the τ138 proteins. These findings are consistent with results from GST pull-down assays (Fig. 4e and Supplementary Fig. 7c). We note that the L469K mutation likely causes a steric clash in the packing of TPR 8 with TPR 7 rather than abolishing a site-specific contact (Fig. 4c). Strikingly, the mutation of residues D468 and L469 has been previously implicated in a loss of binding to TFIIIB subunit Bdp1 (refs 16, 22). We purified full-length, recombinant Bdp1 and performed GST pull-down assays. Bdp1 binding to the TPR array of τ131 is strongly reduced in the D468K or L469K mutants (Fig. 4f). To our knowledge, this is the first time that a loss of interaction by both of these mutations has been probed directly using purified proteins. These results suggest that the binding hotspot for τ138 thus overlaps with that of a binding site for Bdp1.


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)

A binding hotspot on the τ131 TPR array for τ138 and Bdp1.(a) Mapped mutants of the τ131 TPR array (see text for details). (b) Sequence alignment of TPR 8. Identical residues are boxed in brick red, highly conserved in purple, medium conserved in pink and low conserved in white. Coloured arrowheads indicate the five mutated residues. (c) Close-up from the structure of TPR 8. The five residues selected for mutation are displayed as sticks; carbon atoms (grey); oxygen atoms (red). (d) Summary of ITC measurements using indicated τ131 (123–566) point mutants with τ138 (τIR) or τ138 (eWH-τIR). Wild-type (wt) measurements are included for reference. (e) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged τ138 (eWH-τIR). (−) indicates a background control for nonspecific binding of τ138 to the GST-affinity resin. A mixture of purified GST and untagged τ138 was also used as a negative control. Lower gel shows 5% of the input and upper gel shows bound fractions. (f) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged Bdp1. Negative controls and gel format as in e.
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Related In: Results  -  Collection

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f4: A binding hotspot on the τ131 TPR array for τ138 and Bdp1.(a) Mapped mutants of the τ131 TPR array (see text for details). (b) Sequence alignment of TPR 8. Identical residues are boxed in brick red, highly conserved in purple, medium conserved in pink and low conserved in white. Coloured arrowheads indicate the five mutated residues. (c) Close-up from the structure of TPR 8. The five residues selected for mutation are displayed as sticks; carbon atoms (grey); oxygen atoms (red). (d) Summary of ITC measurements using indicated τ131 (123–566) point mutants with τ138 (τIR) or τ138 (eWH-τIR). Wild-type (wt) measurements are included for reference. (e) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged τ138 (eWH-τIR). (−) indicates a background control for nonspecific binding of τ138 to the GST-affinity resin. A mixture of purified GST and untagged τ138 was also used as a negative control. Lower gel shows 5% of the input and upper gel shows bound fractions. (f) GST pull-down assays of purified wild-type (wt) and mutant GST-tagged τ131 (123–566) variants with untagged Bdp1. Negative controls and gel format as in e.
Mentions: Our structure of the τ131 TPR array allows us, for the first time, to map and analyse mutations that have been previously described (Fig. 4a). In detail, mutations that increase Pol III transcription cluster mostly on TPR 2 (ref. 26), those that decrease Pol III transcription spread over TPRs 8–10 (ref. 22) and those that rescue a τ138 temperature-sensitive mutation, map mostly to TPRs 7–8 (ref. 16). Having mapped the critical τ131 interaction region of τ138 to ∼50 amino acids, we next questioned where the τIR binds on the TPR array. Despite extensive efforts, we were unable to obtain structural information of the τ131–τIR complex although both polypeptides form a stable complex during size-exclusion chromatography. Instead, we analysed previously described mutations together with the surface conservation of the TPR array (Supplementary Fig. 3e), to predict where the τIR may bind. We expressed and purified five τ131 point mutants that all cluster on TPR 8 (Fig. 4b). With the exception of residue L469, all of these residues are acidic and surface exposed (Fig. 4c). The purified mutants all eluted at the same volume from a size-exclusion column when compared with the wild type, indicating that the mutations did not cause a destabilization of the proteins (Supplementary Fig. 7a). Using ITC, we determined binding affinities of the mutant τ131 TPR arrays to the τIR and the eWH-τIR (Fig. 4d and Supplementary Fig. 7b). No binding of the mutants D468K and L469K to the τ138 proteins could be detected by ITC. Mutants E472K and E498K showed much weaker binding, while the E497K mutant showed no significant decrease in binding affinity to the τ138 proteins. These findings are consistent with results from GST pull-down assays (Fig. 4e and Supplementary Fig. 7c). We note that the L469K mutation likely causes a steric clash in the packing of TPR 8 with TPR 7 rather than abolishing a site-specific contact (Fig. 4c). Strikingly, the mutation of residues D468 and L469 has been previously implicated in a loss of binding to TFIIIB subunit Bdp1 (refs 16, 22). We purified full-length, recombinant Bdp1 and performed GST pull-down assays. Bdp1 binding to the TPR array of τ131 is strongly reduced in the D468K or L469K mutants (Fig. 4f). To our knowledge, this is the first time that a loss of interaction by both of these mutations has been probed directly using purified proteins. These results suggest that the binding hotspot for τ138 thus overlaps with that of a binding site for Bdp1.

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.


Related in: MedlinePlus