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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.

Show MeSH
Core Factor crosslinking mapLinkage map of crosslinked CF lysine residues. Schematics of each CF subunit showing the predicted domain organization. Purple bars denote lysine positions and the N-terminal amine, while red spheres connected by dashed black lines indicate intra- and inter-molecular crosslinked lysine pairs.
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Figure 3: Core Factor crosslinking mapLinkage map of crosslinked CF lysine residues. Schematics of each CF subunit showing the predicted domain organization. Purple bars denote lysine positions and the N-terminal amine, while red spheres connected by dashed black lines indicate intra- and inter-molecular crosslinked lysine pairs.

Mentions: Incubation of CF with increasing concentrations of BS3 crosslinker showed that all three subunits crosslink with 1:1:1 stoichiometry, resulting in a complex that migrates near the predicted mass sum of the entire complex (~200 KDa) (Fig. 2d). MS analysis detected a total of 71 intramolecular and 39 intermolecular crosslinks within and between the CF subunits, respectively (Fig. 3, Supplementary Table 1, 2). Among them, 43 were identified by both pLink and Nexus; 33 were identified by pLink only and 34 were identified by Nexus only. We detected extensive crosslinking between each of the CF subunits, suggesting an interconnected interaction network where each CF subunit contacts the other two subunits. This conclusion agrees with coexpression studies of different CF subunit combinations in bacteria where all pairwise combinations formed stable heterodimers that can be isolated by His6 affinity chromatography (Fig. 2e). Together, these studies indicate that CF assembles through an extensive network of protein–protein interactions.


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

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

Core Factor crosslinking mapLinkage map of crosslinked CF lysine residues. Schematics of each CF subunit showing the predicted domain organization. Purple bars denote lysine positions and the N-terminal amine, while red spheres connected by dashed black lines indicate intra- and inter-molecular crosslinked lysine pairs.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Core Factor crosslinking mapLinkage map of crosslinked CF lysine residues. Schematics of each CF subunit showing the predicted domain organization. Purple bars denote lysine positions and the N-terminal amine, while red spheres connected by dashed black lines indicate intra- and inter-molecular crosslinked lysine pairs.
Mentions: Incubation of CF with increasing concentrations of BS3 crosslinker showed that all three subunits crosslink with 1:1:1 stoichiometry, resulting in a complex that migrates near the predicted mass sum of the entire complex (~200 KDa) (Fig. 2d). MS analysis detected a total of 71 intramolecular and 39 intermolecular crosslinks within and between the CF subunits, respectively (Fig. 3, Supplementary Table 1, 2). Among them, 43 were identified by both pLink and Nexus; 33 were identified by pLink only and 34 were identified by Nexus only. We detected extensive crosslinking between each of the CF subunits, suggesting an interconnected interaction network where each CF subunit contacts the other two subunits. This conclusion agrees with coexpression studies of different CF subunit combinations in bacteria where all pairwise combinations formed stable heterodimers that can be isolated by His6 affinity chromatography (Fig. 2e). Together, these studies indicate that CF assembles through an extensive network of protein–protein interactions.

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