1. Chromosomes and Gene Expression

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo

  1. Natalia Petrenko
  2. Yi Jin
  3. Koon Ho Wong
  4. Kevin Struhl  Is a corresponding author
  1. Harvard Medical School, United States
  2. University of Macau, China
https://doi.org/10.7554/eLife.28447
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  1. Natalia Petrenko
  2. Yi Jin
  3. Koon Ho Wong
  4. Kevin Struhl
(2017)
Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo
eLife 6:e28447.
https://doi.org/10.7554/eLife.28447
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Abstract

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Data availability

The following data sets were generated
The following previously published data sets were used
    1. Paul et al.
    (2015) Saccharomyces cerevisiae S288c Genome sequencing
    Publicly available via DNA Data Bank of Japan (accession no. SRP047524).
    https://trace.ddbj.nig.ac.jp/DRASearch/study?acc=SRP047524

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. Koon Ho Wong

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

  4. 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, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4181-7856

Funding

National Institutes of Health (GM 30186)

  • Kevin Struhl

Croucher Foundation

  • Koon Ho Wong

University of Macau (MYRG2015-00186-FHS)

  • Koon Ho Wong

University of Macau (MYRG2016-0-0211-FHS)

  • 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

© 2017, 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. Koon Ho Wong
  4. Kevin Struhl
(2017)
Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo
eLife 6:e28447.
https://doi.org/10.7554/eLife.28447

Share this article

https://doi.org/10.7554/eLife.28447

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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.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

    Hans Tobias Gustafsson, Lucas Ferguson ... Oliver J Rando
    Research Article

    Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.