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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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Expression, purification, and chemical crosslinking of active recombinant Core Factor(a) Diagram of the single T7- vector to express recombinant Core Factor (rCF) heterotrimer. (b) Coomassie-stained gel of purified rCF. (c) In vitro transcription assays with a Pol I reporter plasmid and either wild-type (WT) or Δrrn7 yeast extracts in the presence or absence of rCF. Pol I transcripts were detected by primer extension. (d) Chemical crosslinking of rCF. The indicated concentrations of BS3 were incubated with rCF and products were visualized by SDS-PAGE and coomassie-staining. (e) Coexpression and association of pairwise CF subunit combinations. Shown are the inputs of coexpressed proteins and eluates from Ni-agarose pulldowns analyzed by SDS-PAGE and Western blots of the tagged CF subunits.
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Figure 2: Expression, purification, and chemical crosslinking of active recombinant Core Factor(a) Diagram of the single T7- vector to express recombinant Core Factor (rCF) heterotrimer. (b) Coomassie-stained gel of purified rCF. (c) In vitro transcription assays with a Pol I reporter plasmid and either wild-type (WT) or Δrrn7 yeast extracts in the presence or absence of rCF. Pol I transcripts were detected by primer extension. (d) Chemical crosslinking of rCF. The indicated concentrations of BS3 were incubated with rCF and products were visualized by SDS-PAGE and coomassie-staining. (e) Coexpression and association of pairwise CF subunit combinations. Shown are the inputs of coexpressed proteins and eluates from Ni-agarose pulldowns analyzed by SDS-PAGE and Western blots of the tagged CF subunits.

Mentions: We coexpressed three yeast CF subunits from a single vector in bacteria and purified recombinant CF for use in crosslinking analysis. (Fig. 2). Rrn7 and Rrn6, were tagged with His6 for initial purification by Ni-sepharose followed by cation-exchange and size exclusion chromatography, resulting in a pure stoichiometric complex (Fig. 2b). The purified recombinant CF restored Pol I transcription activity of an Δrrn7 yeast extract that is deficient in CF function (Fig. 2c). Adding back recombinant GST-Rrn7 alone could not restore transcription activity (Supplementary Fig. 2).


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

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

Expression, purification, and chemical crosslinking of active recombinant Core Factor(a) Diagram of the single T7- vector to express recombinant Core Factor (rCF) heterotrimer. (b) Coomassie-stained gel of purified rCF. (c) In vitro transcription assays with a Pol I reporter plasmid and either wild-type (WT) or Δrrn7 yeast extracts in the presence or absence of rCF. Pol I transcripts were detected by primer extension. (d) Chemical crosslinking of rCF. The indicated concentrations of BS3 were incubated with rCF and products were visualized by SDS-PAGE and coomassie-staining. (e) Coexpression and association of pairwise CF subunit combinations. Shown are the inputs of coexpressed proteins and eluates from Ni-agarose pulldowns analyzed by SDS-PAGE and Western blots of the tagged CF subunits.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Expression, purification, and chemical crosslinking of active recombinant Core Factor(a) Diagram of the single T7- vector to express recombinant Core Factor (rCF) heterotrimer. (b) Coomassie-stained gel of purified rCF. (c) In vitro transcription assays with a Pol I reporter plasmid and either wild-type (WT) or Δrrn7 yeast extracts in the presence or absence of rCF. Pol I transcripts were detected by primer extension. (d) Chemical crosslinking of rCF. The indicated concentrations of BS3 were incubated with rCF and products were visualized by SDS-PAGE and coomassie-staining. (e) Coexpression and association of pairwise CF subunit combinations. Shown are the inputs of coexpressed proteins and eluates from Ni-agarose pulldowns analyzed by SDS-PAGE and Western blots of the tagged CF subunits.
Mentions: We coexpressed three yeast CF subunits from a single vector in bacteria and purified recombinant CF for use in crosslinking analysis. (Fig. 2). Rrn7 and Rrn6, were tagged with His6 for initial purification by Ni-sepharose followed by cation-exchange and size exclusion chromatography, resulting in a pure stoichiometric complex (Fig. 2b). The purified recombinant CF restored Pol I transcription activity of an Δrrn7 yeast extract that is deficient in CF function (Fig. 2c). Adding back recombinant GST-Rrn7 alone could not restore transcription activity (Supplementary Fig. 2).

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