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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.


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)14 and τ95–τ55 dimerization homologues from S. pombe14. Note that the non-conserved τ55 histidine phosphatase domain (HPD) is omitted13. 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.
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f6: 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)14 and τ95–τ55 dimerization homologues from S. pombe14. Note that the non-conserved τ55 histidine phosphatase domain (HPD) is omitted13. 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.

Mentions: On the basis of limited proteolysis and low-resolution scanning transmission light microscopy, TFIIIC has long been described as a ‘dumb-bell' shaped molecule consisting of two DNA-binding subcomplexes called τA and τB connected by a flexible linker910. More detailed information about the overall architecture of TFIIIC has been lacking. By combining structural information of individual subunits with our crosslinking data sets, we are able to provide a first model of the overall TFIIIC architecture (Fig. 6a). Our XL-MS data, combined with in vitro and in vivo mapping, indicate that the τIR establishes the main link between τA and τB, while the adjacent disordered regions on both sides of the eWH domain in τ138 presumably provide the necessary flexibility for binding variously spaced A box and B box promoters (Figs 2c and 6a). The N-terminal TPR array of τ131 provides a docking platform for the τIR, while C-terminal TPRs interact with the τ95 subunit of τA. Thus, τ131 is crucial for linking the τA and τB subunits. Subunits τ55 and τ95, the two other subunits of the τA subcomplex, share an unexpected structural similarity with the general transcription factor TFIIF14. Subunits τ55 and τ95 dimerize through a triple β-barrel domain consistent with several crosslinks that we observe between their dimerization domains, while the C-terminal DNA-binding domain of τ95 contains a winged helix domain similar to TFIIF Rap30 (ref. 14). In addition to the τIR, our XL-MS data reveal only one other possible link between τA and τB involving the dimerization domain of subunit τ95 and subunit τ138. The importance of this link will have to be substantiated in future studies. In τB, the WD40 propeller subunits τ60 and τ91 have been previously proposed to form a platform for τ138 interaction, thus cooperatively regulating B box binding of τ138 (refs 12, 34). We have observed crosslinks between the τ91 subunit and a disordered region between the third predicted winged helix domain and the eWH domain of τ138, which are not detected when TFIIIC is bound to DNA (Supplementary Fig. 1). In addition, it has been shown that a mutation in the third winged helix domain of τ138 (G349E) strongly reduces the affinity of TFIIIC for DNA18. Considering these different bodies of evidence it is tempting to speculate that this region in τ138 directly recognizes B box sequences.


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 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)14 and τ95–τ55 dimerization homologues from S. pombe14. Note that the non-conserved τ55 histidine phosphatase domain (HPD) is omitted13. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: 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)14 and τ95–τ55 dimerization homologues from S. pombe14. Note that the non-conserved τ55 histidine phosphatase domain (HPD) is omitted13. 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.
Mentions: On the basis of limited proteolysis and low-resolution scanning transmission light microscopy, TFIIIC has long been described as a ‘dumb-bell' shaped molecule consisting of two DNA-binding subcomplexes called τA and τB connected by a flexible linker910. More detailed information about the overall architecture of TFIIIC has been lacking. By combining structural information of individual subunits with our crosslinking data sets, we are able to provide a first model of the overall TFIIIC architecture (Fig. 6a). Our XL-MS data, combined with in vitro and in vivo mapping, indicate that the τIR establishes the main link between τA and τB, while the adjacent disordered regions on both sides of the eWH domain in τ138 presumably provide the necessary flexibility for binding variously spaced A box and B box promoters (Figs 2c and 6a). The N-terminal TPR array of τ131 provides a docking platform for the τIR, while C-terminal TPRs interact with the τ95 subunit of τA. Thus, τ131 is crucial for linking the τA and τB subunits. Subunits τ55 and τ95, the two other subunits of the τA subcomplex, share an unexpected structural similarity with the general transcription factor TFIIF14. Subunits τ55 and τ95 dimerize through a triple β-barrel domain consistent with several crosslinks that we observe between their dimerization domains, while the C-terminal DNA-binding domain of τ95 contains a winged helix domain similar to TFIIF Rap30 (ref. 14). In addition to the τIR, our XL-MS data reveal only one other possible link between τA and τB involving the dimerization domain of subunit τ95 and subunit τ138. The importance of this link will have to be substantiated in future studies. In τB, the WD40 propeller subunits τ60 and τ91 have been previously proposed to form a platform for τ138 interaction, thus cooperatively regulating B box binding of τ138 (refs 12, 34). We have observed crosslinks between the τ91 subunit and a disordered region between the third predicted winged helix domain and the eWH domain of τ138, which are not detected when TFIIIC is bound to DNA (Supplementary Fig. 1). In addition, it has been shown that a mutation in the third winged helix domain of τ138 (G349E) strongly reduces the affinity of TFIIIC for DNA18. Considering these different bodies of evidence it is tempting to speculate that this region in τ138 directly recognizes B box sequences.

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.