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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

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) tDNAGlu 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 xiNET50.
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f1: 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) tDNAGlu 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 xiNET50.

Mentions: While a significant amount of high-resolution structural and functional information exists for some individual TFIIIC subunits121314, a detailed understanding of how the τA and τB subcomplexes are connected and which subunits could act as a flexible linker remains elusive (Fig. 1a). τ95 of τA has been proposed as a linker, based on co-immunoprecipitation experiments that show binding of this subunit to the τB subunits τ138 and τ91 (ref. 15). A genetic study suggests that the two largest TFIIIC subunits, τ138 and τ131, are responsible for connecting τA and τB16. The τ138 subunit is currently the least well-characterized subunit of TFIIIC. Photochemical crosslinking studies support the view that τ138 recognizes the B box promoter of tRNA genes17, as does the mutation of a highly conserved glycine residue (G349E) within τ138 that dramatically reduces the affinity of TFIIIC for tDNA18. Interestingly, this mutation can be suppressed by point mutations within the τ131 subunit, suggesting a direct physical interplay between these two subunits16. τ131 shows the highest sequence conservation of the TFIIIC subunits and is predicted to contain several tetra-trico peptide repeats (TPRs)19. TPRs typically contain 34 amino acids arranged into two antiparallel alpha-helices, with proteins often containing several of these motifs in an array20. This arrangement can lead to an extended, right-handed super-helical structure, which can provide extended binding surfaces for other subunits within a protein assembly21. τ131 fulfills this role within TFIIIC, binding to subunits Brf1 and Bdp1 in a stepwise mechanism to help assemble TFIIIB at tRNA genes, utilizing overlapping sites on an amino-terminal (N-terminal) ‘TPR array'1922232425262728. It has been further hypothesized that the relative positioning of the TPRs and the extended τ131 N terminus mask binding sites within the TPR array, leading to an auto-inhibited τ131 state that must be relieved to allow Brf1 and Bdp1 binding2728. These rate-limiting steps in Pol III PIC formation may be overcome, at least in part, by flexibility within τ131. Indeed, previous studies suggest conformational changes take place within τ131 on the binding of Brf1 and Bdp1 (refs 25, 29). Finally, τ131 has also been shown to bind Pol III subunits Rpc53 and ABC10α3031. Molecular details of the interactions of τ131 with τA or τB subunits have not been reported, although in humans it has been shown that the τ131 orthologue (TFIIIC102) interacts with the τ95 orthologue (TFIIIC63), again requiring the conserved TPR array32.


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)

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) tDNAGlu 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 xiNET50.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490372&req=5

f1: 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) tDNAGlu 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 xiNET50.
Mentions: While a significant amount of high-resolution structural and functional information exists for some individual TFIIIC subunits121314, a detailed understanding of how the τA and τB subcomplexes are connected and which subunits could act as a flexible linker remains elusive (Fig. 1a). τ95 of τA has been proposed as a linker, based on co-immunoprecipitation experiments that show binding of this subunit to the τB subunits τ138 and τ91 (ref. 15). A genetic study suggests that the two largest TFIIIC subunits, τ138 and τ131, are responsible for connecting τA and τB16. The τ138 subunit is currently the least well-characterized subunit of TFIIIC. Photochemical crosslinking studies support the view that τ138 recognizes the B box promoter of tRNA genes17, as does the mutation of a highly conserved glycine residue (G349E) within τ138 that dramatically reduces the affinity of TFIIIC for tDNA18. Interestingly, this mutation can be suppressed by point mutations within the τ131 subunit, suggesting a direct physical interplay between these two subunits16. τ131 shows the highest sequence conservation of the TFIIIC subunits and is predicted to contain several tetra-trico peptide repeats (TPRs)19. TPRs typically contain 34 amino acids arranged into two antiparallel alpha-helices, with proteins often containing several of these motifs in an array20. This arrangement can lead to an extended, right-handed super-helical structure, which can provide extended binding surfaces for other subunits within a protein assembly21. τ131 fulfills this role within TFIIIC, binding to subunits Brf1 and Bdp1 in a stepwise mechanism to help assemble TFIIIB at tRNA genes, utilizing overlapping sites on an amino-terminal (N-terminal) ‘TPR array'1922232425262728. It has been further hypothesized that the relative positioning of the TPRs and the extended τ131 N terminus mask binding sites within the TPR array, leading to an auto-inhibited τ131 state that must be relieved to allow Brf1 and Bdp1 binding2728. These rate-limiting steps in Pol III PIC formation may be overcome, at least in part, by flexibility within τ131. Indeed, previous studies suggest conformational changes take place within τ131 on the binding of Brf1 and Bdp1 (refs 25, 29). Finally, τ131 has also been shown to bind Pol III subunits Rpc53 and ABC10α3031. Molecular details of the interactions of τ131 with τA or τB subunits have not been reported, although in humans it has been shown that the τ131 orthologue (TFIIIC102) interacts with the τ95 orthologue (TFIIIC63), again requiring the conserved TPR array32.

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