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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.


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.
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f5: 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.

Mentions: We tested the binding of τ138 (eWH-τIR) and Bdp1 to τ131 (1–580) (Fig. 5a). Both proteins bind stoichiometrically to this longer τ131 protein, showing that the extended N terminus of τ131 does not inhibit binding. We did not observe binding of Brf1–TBP to τ131 (123–566) under our experimental conditions (data not shown); however, we did detect an interaction using τ131 (1–580; Fig. 5a). The interaction was also observed with the D468K and the L469K mutants of τ131 (1–580) (Fig. 5a). These results indicate that the extended N terminus of τ131 is required for high-affinity binding to Brf1–TBP, and that the principal binding site on τ131 is separate from the hotspot of τ138 and Bdp1. The purified Brf1–TBP contained degradation products of the fusion protein corresponding to truncations lacking the N terminus of Brf1, as confirmed by MS (Supplementary Fig. 8). We observed that these degradation products were not pulled down by τ131 (1–580; Fig. 5a), suggesting that the N terminus of Brf1, which includes the TFIIB-like cyclin repeats, is required for the interaction.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: 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.
Mentions: We tested the binding of τ138 (eWH-τIR) and Bdp1 to τ131 (1–580) (Fig. 5a). Both proteins bind stoichiometrically to this longer τ131 protein, showing that the extended N terminus of τ131 does not inhibit binding. We did not observe binding of Brf1–TBP to τ131 (123–566) under our experimental conditions (data not shown); however, we did detect an interaction using τ131 (1–580; Fig. 5a). The interaction was also observed with the D468K and the L469K mutants of τ131 (1–580) (Fig. 5a). These results indicate that the extended N terminus of τ131 is required for high-affinity binding to Brf1–TBP, and that the principal binding site on τ131 is separate from the hotspot of τ138 and Bdp1. The purified Brf1–TBP contained degradation products of the fusion protein corresponding to truncations lacking the N terminus of Brf1, as confirmed by MS (Supplementary Fig. 8). We observed that these degradation products were not pulled down by τ131 (1–580; Fig. 5a), suggesting that the N terminus of Brf1, which includes the TFIIB-like cyclin repeats, is required for the interaction.

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.