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Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex

Nature Structural & Molecular Biology volume 21, pages 810–816 (2014)Cite this article

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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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Figure 1: Predicted domain organization of Core Factor subunits.
Figure 2: Expression, purification and chemical cross-linking of active recombinant Core Factor.
Figure 3: Linkage map of cross-linked CF lysine residues.
Figure 4: Intramolecular cross-linking within predicted structured domains of Core Factor.
Figure 5: Summary of intermolecular cross-linking, genetic growth phenotypes and biochemical integrity phenotypes.
Figure 6: Molecular model of Core Factor.
Figure 7: CF-TBP cross-links.
Figure 8: Model for the Pol I PIC.

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  • 24 August 2014

    In the version of this article initially published online, there was a mistake in a grant number. The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

We thank members of the S.H. laboratory and of the E. Young (University of Washington) and J. Ranish (Institute for Systems Biology) laboratories for comments throughout the course of the work. We also thank P. Gafkin and the Fred Hutchinson Cancer Research Center proteomics shared resources for assistance with MS and the P. Cramer laboratory (University of Munich) for sharing an early release of their yeast Pol I crystal structure. This work was supported by grants from the US National Institutes of Health (NIH) NIGMS (2RO1GM053451 to S.H. and 2P50 GM076547 to J.R.) and NCI (R21CA175849 to J.R.).

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  1. Bruce A Knutson

    Present address: Present address: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA.,

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  1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Bruce A Knutson & Steven Hahn

  2. Institute for Systems Biology, Seattle, Washington, USA

    Jie Luo & Jeffrey Ranish

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Contributions

B.A.K., J.L., J.R. and S.H. designed the experiments. B.A.K. performed all structural modeling, genetic assays and biochemical experiments. B.A.K. and J.L. performed the cross-linking experiments, and J.L. performed the database searches for the CXMS analysis. B.A.K. and S.H. prepared the manuscript. All authors discussed and interpreted the data and approved the manuscript.

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Correspondence to Steven Hahn.

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Integrated supplementary information

Supplementary Figure 1 Sequence analysis and structural modeling of Core Factor subunits and domains.

(a-c) Immediately above the protein diagrams for Rrn7 (a), Rrn11 (b), and Rrn6 (c) are the structural and functional domain assignments and the 3D structural models. Position of BS3-reactive lysines and amine groups are shown as vertical purple bars within the protein diagrams. Immediately below are the PSIpred secondary structure predictions (Helix, red; Beta-strand, blue; Coil, grey) followed by the DISOPRED disorder predictions shown in green. The scale bar on the y-axis from 0 to 1 indicates the confidence of the predictions from low to high, respectively.

Supplementary Figure 2 Recombinant GST-Rrn7 complements growth in vivo but cannot rescue transcription activity of an rrn7Δ extract in vitro.

(a) RRN7 growth complementation assay. An Δrrn7 yeast strain containing a plasmid that expresses the rDNA locus under control of the GAL7 promoter was transformed with the indicated RRN7 expression constructs and spotted onto glucose complete plates and scored for growth (Wild-type (WT), +++). (b) In vitro transcription assays with a Pol I promoter plasmid and either WT or Δrrn7 yeast extracts in the presence or absence of GST-Rrn7. Pol I transcripts were detected by primer extension.

Supplementary Figure 3 Growth and biochemical phenotypes of Rrn7-deletion mutants.

(a) Growth of yeast strains with indicated mutations in Rrn7. Below the Rrn7 domain map are schematics of the various deletion variants used for the growth assays. Black bars denote regions included in the deletion construct. Each Rrn7 deletion derivative depicted was transformed in strains containing their respective gene deletions and the plasmid pNOY103 that expresses ribosomal RNA under control of the GAL7 promoter. Strains were grown in galactose containing media, streaked onto glucose-containing plates and scored for growth. WT (+++); Lethal (-). Spheres along the top of the domain maps indicate the approximate sites of BS3 crosslinking and are colored accordingly: orange, Rrn11; blue, Rrn6. (b) Assay for CF integrity defects in the Rrn7 deletion derivatives. WT and the indicated deletion variants of Rrn7-Flag were coexpressed in a WT strain that contained epitope tags on the other CF subunits. Each deletion variant was purified from whole-cell extracts and analyzed by Western blotting using the indicated epitope tag antibodies.

Supplementary Figure 4 Growth and biochemical phenotypes of Rrn11-deletion mutants.

(a) Growth of yeast strains with indicated mutations in Rrn11. Growth assays were as described in Supplementary Figure 3a. Spheres along the top of the domain maps indicate the approximate sites of BS3 crosslinking and are colored accordingly: green, Rrn7; blue, Rrn6. (b) Assay for CF integrity defects in the Rrn11 deletion derivatives. WT and indicated deletion variants of Rrn11-Myc were coexpressed in a WT strain that contained unique epitope tags on the other CF subunits. IP and Western blot analysis were performed as in Supplementary Figure 3b.

Supplementary Figure 5 Growth and biochemical phenotypes of Rrn6-deletion mutants.

(a,c) Growth of yeast strains with indicated mutations in Rrn6. Growth assay were done as described in Supplementary Figure 3a. Spheres along the top of the domain maps indicate the approximate sites of BS3 crosslinking and are colored accordingly: green, Rrn7; orange, Rrn11. N.D., not determined. (b,d) Assay for CF integrity defects in the Rrn6 deletion derivatives. WT and indicated deletion variants of Rrn6-Myc were coexpressed in a WT strain that contained unique epitope tags on the other CF subunits. IP and Western blot analysis were performed as in Supplementary Figure 3b.

Supplementary Figure 6 Recruitment of CF-deletion variants to the rDNA promoter.

(a) Location of PCR primer sets (red bars) within the 35S rDNA locus and Pol I promoter. Promoter, Pro; External Transcribed Spacer, ETS. (b-d) Strains containing either WT Rrn7 (b), Rrn11 (c), Rrn6 (d), and the indicated CF subunit derivatives were crosslinked with formaldehyde and analyzed by chromatin IP and qPCR using the indicated primers sets. Averages of biological duplicate experiments are expressed relative to WT, which was set at 1.0. Error bars indicate standard deviations. An asterisk denotes lethal CF mutants that retain complex integrity.

Supplementary Figure 7 Molecular topology of Core Factor and position of the Rrn7 CTD.

(a) Topological linkage map showing all the observed intermolecular and interdomain crosslinks between the CF subunit domains. 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 black lines. (b) Lysine residue Cα positions within the CF model that crosslink to the Rrn7-CTD are shown as green spheres connected by black lines to numbers indicating the crosslinked Rrn7-CTD lysine residue. Rrn7-CTD lysines K329 and K360 reside in the helical segment H1 and the linker between H2 and H3, respectively. Rrn7-CTD lysine K457 lies within the linker between H5 and H6, while the K473 and K495, 499, and 506 reside in H6 and H7, respectively. The green dashed outline indicates the approximate location of the Rrn7-CTD, suggested by the mapped Rrn7-CTD crosslink positions.

Supplementary Figure 8 Molecular topology and architecture of CF–TBP complex.

(a) Linkage map of crosslinked CF-TBP lysine residues. Schematics of TBP and each CF subunit showing their known and 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 within TBP or between TBP and CF. (b) Intramolecular crosslinks within the TBP structure (1YTB). Non-crosslinked lysine Cα atoms are depicted as spheres. Cα atoms of crosslinked lysine pairs are depicted as red spheres connected by black lines (d-e) Model of the CF-TBP-DNA complex shown without (d) or with TBP and DNA (e). (c-f) Calculated Cα-Cα distances between crosslinked lysine pairs within TBP (c) or between CF subunits and TBP (f). Dashed grey line denotes the theoretical maximum crosslinking distance for BS3 of 30 Å.

Supplementary Figure 9 Full images of cropped panels.

The figure number and corresponding panel are indicated at the top of each image.

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Supplementary Figures 1–9 and Supplementary Tables 1–5 (PDF 6211 kb)

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Knutson, B., Luo, J., Ranish, J. et al. Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex. Nat Struct Mol Biol 21, 810–816 (2014). https://doi.org/10.1038/nsmb.2873

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