In vitro analysis of RNA polymerase II elongation complex dynamics
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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).”
From RNA polymerase II speed: a key player in controlling and adapting transcriptome composition (2021) · cites this paper as Joo et al, 2019
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“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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Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genesPreprint
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Dynamics of TFIIH and Spt4/5 during the transition from transcription initiation to elongationPreprint
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Self-citationDynamics of TFIIH and Spt4/5 during the transition from transcription initiation to elongation
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Self-citation
- 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
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2019-03-07
- Volume
- 33
- Issue
- 9-10
- Pages
- 578-589
- ISSN
- 0890-9369 (ISSN-L)
- 1549-5477
- PMC
- PMC6499329
- 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
- hybrid
- doi.org
- 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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