Limits...
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

The τ131 TPR array interacts with high affinity to a central region of τ138.ITC measurement using purified (a) τ138 (546–693) and τ131 (123–566); (b) τ138 (641–693) and τ131 (123–566); Calculated Kd values and stoichiometry (N) are indicated. 15 μM of τ138 was used in the cell and 150 μM τ131 was used in the syringe in each case. (c) Viability of τ138 deletion mutants in vivo determined by the spot assay. A yeast strain carrying the plasmid pOL49 was transformed with the plasmids pRS415 ΔCEN τ138 and pRS415 ΔCEN Δ546-693 or Δ641-693 or Δ546-641 and plated on SC-URA-LEU medium. Serial 10-fold dilutions of all strains were spotted on SC-LEU and 5-fluoroorotic acid medium and were incubated for 72 h at 30 °C (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490372&req=5

f3: The τ131 TPR array interacts with high affinity to a central region of τ138.ITC measurement using purified (a) τ138 (546–693) and τ131 (123–566); (b) τ138 (641–693) and τ131 (123–566); Calculated Kd values and stoichiometry (N) are indicated. 15 μM of τ138 was used in the cell and 150 μM τ131 was used in the syringe in each case. (c) Viability of τ138 deletion mutants in vivo determined by the spot assay. A yeast strain carrying the plasmid pOL49 was transformed with the plasmids pRS415 ΔCEN τ138 and pRS415 ΔCEN Δ546-693 or Δ641-693 or Δ546-641 and plated on SC-URA-LEU medium. Serial 10-fold dilutions of all strains were spotted on SC-LEU and 5-fluoroorotic acid medium and were incubated for 72 h at 30 °C (n=3).

Mentions: We next wanted to characterize further the central region of τ138 (546–693) and understand which regions were crucial for interaction with the TPR array. We hypothesized that the inner groove of the TPR array may accommodate the predicted central winged helix domain of τ138 (Fig. 2c). Surface analysis of the TPR array shows that the inner groove is lined with patches of conserved, often acidic surface residues that may bind to the predicted basic surface residues of τ138 (Supplementary Figs 3c,d,e and 4). We first solved the structure of the central domain of τ138 (546–641) using the sulphur-SAD technique (Table 1, Supplementary Table 5, Supplementary Fig. 5a). The 1.4 Å crystal structure (Rwork/Rfree of 17.5%/19.8%) of this domain reveals a canonical winged helix domain that contains an additional C-terminal helix (Fig. 2d). This first structurally characterized part of τ138 thus represents an ‘extended' winged helix (eWH) domain35. The eWH domain is moderately well conserved from yeast to human (Supplementary Fig. 5b,c,d), and contains basic patches that could also suggest a role in binding nucleic acids (Supplementary Fig. 5e). However, we only detect very weak unspecific binding of the eWH domain to single and double-stranded DNA (Supplementary Fig. 5f). Isothermal titration calorimetry (ITC) did not detect an interaction between the eWH and the TPR array (Supplementary Fig. 6a). However, when we tested a construct that contained the eWH domain with additional residues at the C terminus (546–693), we observed a Kd for the interaction with the TPR array of ∼100 nM (Fig. 3a). A similar high-affinity interaction (∼80 nM) could be measured with only the unstructured region of τ138 (641–693) (Fig. 3b). Removal of residues 682–693 lowered the Kd of the interaction to ∼2.6 μM (Supplementary Fig. 6b), yet the region 641–681 was still essential to ensure the high-affinity interaction as tested peptides of 681–693 did not interact with the TPR array by ITC (data not shown). We thus concluded that the region 641–693 of τ138, hereafter referred to as ‘τ131-Interaction Region (τIR)', is necessary and sufficient to bind the TPR array of τ131.


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)

The τ131 TPR array interacts with high affinity to a central region of τ138.ITC measurement using purified (a) τ138 (546–693) and τ131 (123–566); (b) τ138 (641–693) and τ131 (123–566); Calculated Kd values and stoichiometry (N) are indicated. 15 μM of τ138 was used in the cell and 150 μM τ131 was used in the syringe in each case. (c) Viability of τ138 deletion mutants in vivo determined by the spot assay. A yeast strain carrying the plasmid pOL49 was transformed with the plasmids pRS415 ΔCEN τ138 and pRS415 ΔCEN Δ546-693 or Δ641-693 or Δ546-641 and plated on SC-URA-LEU medium. Serial 10-fold dilutions of all strains were spotted on SC-LEU and 5-fluoroorotic acid medium and were incubated for 72 h at 30 °C (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490372&req=5

f3: The τ131 TPR array interacts with high affinity to a central region of τ138.ITC measurement using purified (a) τ138 (546–693) and τ131 (123–566); (b) τ138 (641–693) and τ131 (123–566); Calculated Kd values and stoichiometry (N) are indicated. 15 μM of τ138 was used in the cell and 150 μM τ131 was used in the syringe in each case. (c) Viability of τ138 deletion mutants in vivo determined by the spot assay. A yeast strain carrying the plasmid pOL49 was transformed with the plasmids pRS415 ΔCEN τ138 and pRS415 ΔCEN Δ546-693 or Δ641-693 or Δ546-641 and plated on SC-URA-LEU medium. Serial 10-fold dilutions of all strains were spotted on SC-LEU and 5-fluoroorotic acid medium and were incubated for 72 h at 30 °C (n=3).
Mentions: We next wanted to characterize further the central region of τ138 (546–693) and understand which regions were crucial for interaction with the TPR array. We hypothesized that the inner groove of the TPR array may accommodate the predicted central winged helix domain of τ138 (Fig. 2c). Surface analysis of the TPR array shows that the inner groove is lined with patches of conserved, often acidic surface residues that may bind to the predicted basic surface residues of τ138 (Supplementary Figs 3c,d,e and 4). We first solved the structure of the central domain of τ138 (546–641) using the sulphur-SAD technique (Table 1, Supplementary Table 5, Supplementary Fig. 5a). The 1.4 Å crystal structure (Rwork/Rfree of 17.5%/19.8%) of this domain reveals a canonical winged helix domain that contains an additional C-terminal helix (Fig. 2d). This first structurally characterized part of τ138 thus represents an ‘extended' winged helix (eWH) domain35. The eWH domain is moderately well conserved from yeast to human (Supplementary Fig. 5b,c,d), and contains basic patches that could also suggest a role in binding nucleic acids (Supplementary Fig. 5e). However, we only detect very weak unspecific binding of the eWH domain to single and double-stranded DNA (Supplementary Fig. 5f). Isothermal titration calorimetry (ITC) did not detect an interaction between the eWH and the TPR array (Supplementary Fig. 6a). However, when we tested a construct that contained the eWH domain with additional residues at the C terminus (546–693), we observed a Kd for the interaction with the TPR array of ∼100 nM (Fig. 3a). A similar high-affinity interaction (∼80 nM) could be measured with only the unstructured region of τ138 (641–693) (Fig. 3b). Removal of residues 682–693 lowered the Kd of the interaction to ∼2.6 μM (Supplementary Fig. 6b), yet the region 641–681 was still essential to ensure the high-affinity interaction as tested peptides of 681–693 did not interact with the TPR array by ITC (data not shown). We thus concluded that the region 641–693 of τ138, hereafter referred to as ‘τ131-Interaction Region (τIR)', is necessary and sufficient to bind the TPR array of τ131.

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