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Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex.

Knutson BA, Luo J, Ranish J, Hahn S - Nat. Struct. Mol. Biol. (2014)

Bottom Line: The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1.Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex.We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2].

ABSTRACT
Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking-derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

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Model for the Pol I PIC(a-c) The Pol I PIC model is based on current Pol II/III PIC models. The following structures were used to create the model: Pol I (Pdb, 4C2M)15, Rrn3 (Pdb, 3TJ1)12, A49-WH (Pdb, 3NFI)19, TBP (Pdb, 1YTB)55. (a) The Pol I PIC model creates an empty canyon (circled in red) between the Rrn7-CR, TBP, and Rrn3. Cα atoms of lysines within the Rrn7-CR and TBP that crosslink to the Rrn6-HB are shown as red colored spheres (b) Positioning of the Rrn6-HB into the empty canyon guided by crosslinking restraints between the Rrn6-HB and Rrn7-CR domains and TBP. Crosslinked lysine pairs are shown as red spheres connected by black lines, and the Cα-Cα distances between each crosslinked lysine pair are below 23 Å. (c) Surface representation of the Pol I PIC model including the entire CF domain model.
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Figure 8: Model for the Pol I PIC(a-c) The Pol I PIC model is based on current Pol II/III PIC models. The following structures were used to create the model: Pol I (Pdb, 4C2M)15, Rrn3 (Pdb, 3TJ1)12, A49-WH (Pdb, 3NFI)19, TBP (Pdb, 1YTB)55. (a) The Pol I PIC model creates an empty canyon (circled in red) between the Rrn7-CR, TBP, and Rrn3. Cα atoms of lysines within the Rrn7-CR and TBP that crosslink to the Rrn6-HB are shown as red colored spheres (b) Positioning of the Rrn6-HB into the empty canyon guided by crosslinking restraints between the Rrn6-HB and Rrn7-CR domains and TBP. Crosslinked lysine pairs are shown as red spheres connected by black lines, and the Cα-Cα distances between each crosslinked lysine pair are below 23 Å. (c) Surface representation of the Pol I PIC model including the entire CF domain model.

Mentions: The macromolecular architectures of the Pol II and III PICs have been deduced at varying degrees of resolution and may give clues to the architecture of the Pol I PIC. A common theme between the Pol II and III PIC architectures is the position of the TFIIB and Brf1 cyclin fold repeats and their position with respect to Pol, TBP and promoter DNA. Using the Pol II and III PIC models as a guide12,48-53, we created a Pol I PIC model containing the Pol I and Rrn3 structures with TBP, DNA, and our Rrn7 homology model. A striking feature of this Pol I PIC model is an empty canyon between Rrn7 and Rrn3 where Pol I forms the floor of the canyon, and the Rrn7 cyclin folds, DNA, and TBP form one wall, and Rrn3 forms the opposing wall (Fig. 8a). Rrn7 lysines within the cyclin domain that crosslink to the Rrn6-HB domain are accessible and face the empty canyon pointed toward Rrn3, and the Rrn6-HB can be positioned within the canyon and satisfy the BS3 crosslinking distance constraints (Fig. 8b). This model explains how Rrn6 mediates simultaneous interactions with Rrn7, Rrn3, and TBP.


Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex.

Knutson BA, Luo J, Ranish J, Hahn S - Nat. Struct. Mol. Biol. (2014)

Model for the Pol I PIC(a-c) The Pol I PIC model is based on current Pol II/III PIC models. The following structures were used to create the model: Pol I (Pdb, 4C2M)15, Rrn3 (Pdb, 3TJ1)12, A49-WH (Pdb, 3NFI)19, TBP (Pdb, 1YTB)55. (a) The Pol I PIC model creates an empty canyon (circled in red) between the Rrn7-CR, TBP, and Rrn3. Cα atoms of lysines within the Rrn7-CR and TBP that crosslink to the Rrn6-HB are shown as red colored spheres (b) Positioning of the Rrn6-HB into the empty canyon guided by crosslinking restraints between the Rrn6-HB and Rrn7-CR domains and TBP. Crosslinked lysine pairs are shown as red spheres connected by black lines, and the Cα-Cα distances between each crosslinked lysine pair are below 23 Å. (c) Surface representation of the Pol I PIC model including the entire CF domain model.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219626&req=5

Figure 8: Model for the Pol I PIC(a-c) The Pol I PIC model is based on current Pol II/III PIC models. The following structures were used to create the model: Pol I (Pdb, 4C2M)15, Rrn3 (Pdb, 3TJ1)12, A49-WH (Pdb, 3NFI)19, TBP (Pdb, 1YTB)55. (a) The Pol I PIC model creates an empty canyon (circled in red) between the Rrn7-CR, TBP, and Rrn3. Cα atoms of lysines within the Rrn7-CR and TBP that crosslink to the Rrn6-HB are shown as red colored spheres (b) Positioning of the Rrn6-HB into the empty canyon guided by crosslinking restraints between the Rrn6-HB and Rrn7-CR domains and TBP. Crosslinked lysine pairs are shown as red spheres connected by black lines, and the Cα-Cα distances between each crosslinked lysine pair are below 23 Å. (c) Surface representation of the Pol I PIC model including the entire CF domain model.
Mentions: The macromolecular architectures of the Pol II and III PICs have been deduced at varying degrees of resolution and may give clues to the architecture of the Pol I PIC. A common theme between the Pol II and III PIC architectures is the position of the TFIIB and Brf1 cyclin fold repeats and their position with respect to Pol, TBP and promoter DNA. Using the Pol II and III PIC models as a guide12,48-53, we created a Pol I PIC model containing the Pol I and Rrn3 structures with TBP, DNA, and our Rrn7 homology model. A striking feature of this Pol I PIC model is an empty canyon between Rrn7 and Rrn3 where Pol I forms the floor of the canyon, and the Rrn7 cyclin folds, DNA, and TBP form one wall, and Rrn3 forms the opposing wall (Fig. 8a). Rrn7 lysines within the cyclin domain that crosslink to the Rrn6-HB domain are accessible and face the empty canyon pointed toward Rrn3, and the Rrn6-HB can be positioned within the canyon and satisfy the BS3 crosslinking distance constraints (Fig. 8b). This model explains how Rrn6 mediates simultaneous interactions with Rrn7, Rrn3, and TBP.

Bottom Line: The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1.Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex.We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2].

ABSTRACT
Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking-derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

Show MeSH
Related in: MedlinePlus