Limits...
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
CF-TBP crosslinksTopological linkage map showing all the observed intermolecular and interdomain crosslinks between the CF subunit domains and TBP. Non-crosslinked lysine Cα atoms are depicted as spheres in the same color as the domain. Crosslinked lysine pairs are shown as red spheres connected by grey or black lines. Gray lines denote crosslinks between CF domains and black lines denotes crosslinks between CF and TBP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219626&req=5

Figure 7: CF-TBP crosslinksTopological linkage map showing all the observed intermolecular and interdomain crosslinks between the CF subunit domains and TBP. Non-crosslinked lysine Cα atoms are depicted as spheres in the same color as the domain. Crosslinked lysine pairs are shown as red spheres connected by grey or black lines. Gray lines denote crosslinks between CF domains and black lines denotes crosslinks between CF and TBP.

Mentions: Little is known about the interactions between TBP and the Pol I GTFs. Previous protein interaction assays suggest that each CF subunit can independently interact with TBP, but that the strongest TBP interactions are with Rrn6 45,46. To examine the interactions between TBP and CF, we crosslinked CF-TBP complexes to identify regions of these factors in close proximity. We detected 23 intermolecular crosslinks between TBP and CF subunits that clustered into several essential domains within the CF subunits (Supplementary Table 3, Fig. 7). We also detected 23 intramolecular crosslinks within TBP (Supplementary Table 3, Fig. 7). We mapped seven of these intramolecular crosslinks to the TBP structure and all fall within the BS3 distance constraints (Supplementary Fig. 8b,c). Crosslinks between the TBP N-terminus and CF subunits are less informative since several reports suggest that the TBP N-terminus is not required for rDNA transcription nor CF and SL1 interaction47.


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

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

CF-TBP crosslinksTopological linkage map showing all the observed intermolecular and interdomain crosslinks between the CF subunit domains and TBP. Non-crosslinked lysine Cα atoms are depicted as spheres in the same color as the domain. Crosslinked lysine pairs are shown as red spheres connected by grey or black lines. Gray lines denote crosslinks between CF domains and black lines denotes crosslinks between CF and TBP.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: CF-TBP crosslinksTopological linkage map showing all the observed intermolecular and interdomain crosslinks between the CF subunit domains and TBP. Non-crosslinked lysine Cα atoms are depicted as spheres in the same color as the domain. Crosslinked lysine pairs are shown as red spheres connected by grey or black lines. Gray lines denote crosslinks between CF domains and black lines denotes crosslinks between CF and TBP.
Mentions: Little is known about the interactions between TBP and the Pol I GTFs. Previous protein interaction assays suggest that each CF subunit can independently interact with TBP, but that the strongest TBP interactions are with Rrn6 45,46. To examine the interactions between TBP and CF, we crosslinked CF-TBP complexes to identify regions of these factors in close proximity. We detected 23 intermolecular crosslinks between TBP and CF subunits that clustered into several essential domains within the CF subunits (Supplementary Table 3, Fig. 7). We also detected 23 intramolecular crosslinks within TBP (Supplementary Table 3, Fig. 7). We mapped seven of these intramolecular crosslinks to the TBP structure and all fall within the BS3 distance constraints (Supplementary Fig. 8b,c). Crosslinks between the TBP N-terminus and CF subunits are less informative since several reports suggest that the TBP N-terminus is not required for rDNA transcription nor CF and SL1 interaction47.

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