1. Chromosomes and Gene Expression
  2. Genetics and Genomics

Requirements for RNA polymerase II preinitiation complex formation in vivo

  1. Natalia Petrenko
  2. Yi Jin
  3. Liguo Dong
  4. Koon Ho Wong  Is a corresponding author
  5. Kevin Struhl  Is a corresponding author
  1. Harvard Medical School, United States
  2. University of Macau, Macau
https://doi.org/10.7554/eLife.43654
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  1. Natalia Petrenko
  2. Yi Jin
  3. Liguo Dong
  4. Koon Ho Wong
  5. Kevin Struhl
(2019)
Requirements for RNA polymerase II preinitiation complex formation in vivo
eLife 8:e43654.
https://doi.org/10.7554/eLife.43654
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Abstract

Transcription by RNA polymerase II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the promoter. In vitro, some GTFs are essential for transcription, whereas others are not required under certain conditions. PICs are stable in the absence of nucleotide triphosphates, and subsets of GTFs can form partial PICs. By depleting individual GTFs in yeast cells, we show that all GTFs are essential for TBP binding and transcription, suggesting that partial PICs do not exist at appreciable levels in vivo. Depletion of FACT, a histone chaperone that travels with elongating Pol II, strongly reduces PIC formation and transcription. In contrast, TBP-associated factors (TAFs) contribute to transcription of most genes, but TAF-independent transcription occurs at substantial levels, preferentially at promoters containing TATA elements. PICs are absent in cells deprived of uracil, and presumably UTP, suggesting that transcriptionally inactive PICs are removed from promoters in vivo.

Data availability

Sequencing data has been deposited in GEO under the accession number GSE122734

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Natalia Petrenko

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.

  2. Yi Jin

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.

  3. Liguo Dong

    Faculty of Health Sciences, University of Macau, Macau, Macau
    Competing interests
    No competing interests declared.

  4. Koon Ho Wong

    Faculty of Health Sciences, University of Macau, Macau, Macau
    For correspondence
    KoonHoWong@umac.mo
    Competing interests
    No competing interests declared.

  5. Kevin Struhl

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    kevin@hms.harvard.edu
    Competing interests
    Kevin Struhl, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4181-7856

Funding

National Institutes of Health (GM 30186)

  • Natalia Petrenko
  • Yi Jin
  • Koon Ho Wong
  • Kevin Struhl

Universidade de Macau (MYRG2015-00186 FHS)

  • Liguo Dong
  • Koon Ho Wong

Croucher Foundation

  • Koon Ho Wong

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Petrenko et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Natalia Petrenko
  2. Yi Jin
  3. Liguo Dong
  4. Koon Ho Wong
  5. Kevin Struhl
(2019)
Requirements for RNA polymerase II preinitiation complex formation in vivo
eLife 8:e43654.
https://doi.org/10.7554/eLife.43654

Share this article

https://doi.org/10.7554/eLife.43654

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Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Comparison of transcriptional initiation by RNA polymerase II across eukaryotic species

    Natalia Petrenko, Kevin Struhl
    Research Article Updated

    The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.