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Identification of TFB5, a new component of general transcription and DNA repair factor IIH

Nature Genetics volume 36, pages 707–713 (2004)Cite this article

Abstract

We previously described the use of quantitative proteomics to study macromolecular complexes1. Applying the method to analyze a yeast RNA polymerase II preinitiation complex, we identified a new 8-kDa protein, encoded by the uncharacterized open reading frame YDR079c-a, as a potential new component of the preinitiation complex. Here we show that YDR079c-a is a bona fide component of polymerase II preinitiation complexes and investigate its role in transcription. YDR079c-a is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL genes. In addition, YDR079c-a is a core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to a promoter. Yeast lacking YDR079c-a grow slowly, and, like strains carrying mutations in core TFIIH subunits, are sensitive to ultraviolet radiation. YDR079c-a is conserved throughout evolution, and mutations in the human ortholog account for a DNA repair–deficient form of the tricothiodystrophy disorder called TTD-A2. The identification of a new, evolutionarily conserved, core TFIIH subunit is essential for our understanding of TFIIH function in transcription, DNA repair and human disease.

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Figure 1: Identification of YDR079c-a by quantitative proteomic analysis of promoter-DNA purified Pol II PICs.
Figure 2: Growth phenotypes of the YDR079c-a deletion strain.
Figure 3: YDR079c-a is required for normal expression of GAL genes and is recruited to actively transcribed genes.
Figure 4: Quantitative proteomic analysis of YDR079c-a-FLAG immunoprecipitates.
Figure 5: YDR079c-a is a subunit of core TFIIH.
Figure 6: YDR079c-a is required for stable recruitment of TFIIH to a promoter but not for stability of TFIIH subunits.

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Acknowledgements

We thank P. Mallick and B. Marzolf for help with data analysis, E. Schiebel for the pYM plasmids, T. Tsukiyama for pUG6 and p3FLAGKanMX plasmids, S. Buratowski for antibodies to TFB3 antibodies and yeast strains and Y. Chi, T. Krumm and N. Yudkovsky for critical reading of the manuscript. This work was supported by grants from the US National Cancer Institute and the National Institutes of Health Research Resource Center, by federal funds from National Heart, Lung, and Blood Institute, by a US National Institutes of Health contract and by a postdoctoral fellowship from the National Institutes of Health to J.A.R. Partial funding for this work came from a gift from Merck and Company to the Institute for Systems Biology.

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Authors and Affiliations

  1. Institute for Systems Biology, 1441 North 34th Street, Seattle, 98103-8904, Washington, USA

    Jeffrey A Ranish, Yu Lu, Eugene C Yi, Xiao-jun Li, Jimmy Eng & Ruedi Aebersold

  2. The Howard Hughes Medical Institute and Division of Basic Sciences, The Fred Hutchinson Cancer Research Center, Seattle, 98109, Washington, USA

    Steven Hahn

  3. Department of Chemistry, University of Washington, Seattle, 98195, Washington, USA

    Yu Lu

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  1. Jeffrey A Ranish
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Correspondence to Jeffrey A Ranish.

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Supplementary information

Supplementary Fig. 1

Results of the P-BLAST search with the YDR079c-a proptein sequence (Feb. 21, 2003). (PDF 92 kb)

Supplementary Fig. 2

YDR079c-a is required for efficient transcription initiation in vitro. (PDF 149 kb)

Supplementary Table 1

Results of DNA microarray analysis of poly(A)+ RNA from wild-type and YDR079c-a deletion strains. (XLS 3134 kb)

Supplementary Table 2

Quantitative proteomic analysis of YDR079c-a-FLAG immunoprecipitation. (XLS 31 kb)

Supplementary Table 3

Yeast strains used in this study. (XLS 10 kb)

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Ranish, J., Hahn, S., Lu, Y. et al. Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nat Genet 36, 707–713 (2004). https://doi.org/10.1038/ng1385

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