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

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.

Figure 1. XL-MS of purified, endogenous TFIIIC reveals a link between τA and τB.

(a) Schematic representation of the six subunits of S. cerevisiae TFIIIC. The amino-acid lengths of the subunits are labelled at the C terminus. Domains of which crystal structures are available are highlighted. τ55 and τ95 Dim.=τ55 and τ95 dimerization domains; DBD, DNA-binding domain; HPD, histidine phosphatase domain. The tetra-trico peptide (TPR) array of τ131 and the eWH domain of τ138 are included, see text for details. Additional predicted structural regions of τ131 and τ138 are highlighted in grey. HMG, high mobility group box domain; WH=winged helix. (b) Analytical size-exclusion chromatography profile of TFIIIC using a Superose 6 10/300 column (GE Healthcare). Known molecular weight standards at 670 and 158 kDa are indicated. Inset, Coomassie-stained SDS–PAGE gel of an elution peak fraction. (c) EMSA experiment of TFIIIC bound to a double-stranded (ds) 66 base-pair (bp) tDNA^Glu oligonucleotide. C, control (no TFIIIC added). (d) Crosslinking map of TFIIIC. TFIIIC subunits are represented as in a with internal vertical lines representing 100 amino-acid markers. Intra crosslinks are depicted by arcs that connect residues within the same subunit. Inter crosslinks are depicted by lines which connect residues within different subunits. Image produced using xiNET.

Figure 2. 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.

Figure 3. 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 K_d 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).

Figure 4. 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.

Figure 5. Defining the overlap between TFIIIB and τ138 binding to τ131.

(a) GST pull-down assays of purified wild-type (wt) GST-tagged τ131 (1–580) with untagged τ138 (eWH-τIR) and Bdp1, and purified wild-type (wt) and mutant GST-tagged τ131 (1–580) variants with Brf1–TBP. (−) indicates a background control for nonspecific binding of Brf1–TBP to the GST-affinity resin. A mixture of purified GST and untagged Brf1–TBP was also used as a negative control. Lower gel shows 5% of the input and upper gel shows bound fractions. An * in the input gel indicates degradation products of Brf1–TBP. (b) GST pull-down competition assays of purified wild-type (wt) GST-tagged τ131 (1–580) with untagged τ138 (eWH-τIR) and Bdp1. GST-τ131 was preincubated with τ138 before addition of the indicated molar excess of Bdp1. (−) indicates a control experiment where no Bdp1 was added. Gel format as in a. (c) GST pull-down competition assays of purified wild-type (wt) GST-tagged τ131 (1–580) with untagged τ138 (eWH-τIR) and Brf1–TBP. GST-τ131 was preincubated with τ138 before addition of the indicated molar excess of Brf1–TBP. (−) indicates a control experiment where no Brf1–TBP was added. Gel format as in a. An * in the input gel indicates degradation products of Brf1–TBP.

Figure 6. The role of τ131 in Pol III PIC formation.

(a) Our current view of the arrangement of τA and τB subunits within TFIIIC based on interaction studies, crystal structures and crosslinking. Structures from this study are included, as well as previous structures with PDB codes indicated: the WD40 dimer structure of τ60–τ91 (ref. 12), and the τ95 DNA-binding domain (DBD) and τ95–τ55 dimerization homologues from S. pombe. Note that the non-conserved τ55 histidine phosphatase domain (HPD) is omitted. The extended N terminus and C-terminal TPRs of τ131 are indicated schematically. Predicted structural regions of τ138 are also indicated, including the G349E mutation. The disordered N terminus of τ91 is included schematically. (b) Model indicating two stages of PIC formation. In the first stage, recruitment of Brf1 to the PIC requires the extended N terminus of τ131 (red curve) and the N-terminal TPRs of the TPR array (highlighted in blue). TBP makes interactions with Brf1 and the τ60 subunit of τB. The link between τ131 and τ138 is maintained. In the second stage, the recruitment of Bdp1 involves conformational changes in the arms of the TPR array. τIR is displaced and the τA–τB link is altered, possibly leading to the disassembly of TFIIIC. TFIIIB is now assembled and recruits Pol III, together with τ131, for transcription.