PreprintCited by 44Genomics and Chromatin DynamicsRNA Research and SplicingNuclear Structure and Function

In vitro analysis of RNA polymerase II elongation complex dynamics

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Joo, Yoo Jin, Ficarro, Scott B., Chun, Yujin, Marto, Jarrod A., Buratowski, Stephen·2019·Genes & Development

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Abstract

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4–Spt5, Paf1C, Spt6–Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

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  • RNA Pol II CTD phosphorylation patterns and the factors recognizing them change as a function of post-initiation time rather than distance elongated.

    “Interestingly, in vitro transcription analyses have shown that the RNA Pol II CTD phosphorylation patterns and the factors recognizing them change as a function of post‐initiation time rather than distance elongated (Joo et al, 2019).”

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Authors
Joo, Y. J., Ficarro, S. B., Chun, Y., Marto, J. A., & Buratowski, S.
Affiliations
Harvard University (US)
Corresponding author
Buratowski, Stephen
Venue
Genes & Development
Published
2019-03-07
Volume
33
Issue
9-10
Pages
578-589
ISSN
  • 0890-9369 (ISSN-L)
  • 1549-5477
OpenAlex
W2907689105
Cited by
44
References
87
Citations by year
2026: 5 · 2025: 8 · 2024: 4 · 2023: 5 · 2022: 7 · 2021: 7 · 2020: 5 · 2019: 3
Type
posted content
Language
en
Open access
Licence
cc-by-nc
Locations
  • Genes & Development (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • Dana Farber Cancer Institute/Blaise Proteomics Center
  • National Institutes of Health (GM46498, GM56663)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
Keywords
transcription elongation, TFIIF, Paf1 complex, ELF1, capping enzyme, cap-binding complex, Set2
MeSH terms
Enzyme Activation, Peptide Elongation Factors, Phosphorylation, Protein Kinases, RNA Polymerase II, Saccharomyces cerevisiae, Cyclin-Dependent Kinases, Saccharomyces cerevisiae Proteins

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