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Related Experiment Videos

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

P A Kolodziej1, N Woychik, S M Liao

  • 1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.

Molecular and Cellular Biology
|May 1, 1990
PubMed
Summary

Researchers precisely defined the subunit composition and phosphorylation of yeast RNA polymerase II (RPB). They found three subunits (RPB1, RPB2, RPB6) are phosphorylated in vivo, with RPB1 being partially phosphorylated.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Yeast Genetics

Background:

  • RNA polymerase II is crucial for eukaryotic gene transcription.
  • Understanding its subunit composition and post-translational modifications is key to deciphering gene regulation.

Purpose of the Study:

  • To precisely determine the subunit composition, stoichiometry, and phosphorylation status of RNA polymerase II in Saccharomyces cerevisiae.
  • To identify which subunits are phosphorylated in vivo and to characterize the phosphorylation of RPB1.

Main Methods:

  • Epitope tagging of the RPB3 gene to enable immunoprecipitation of RNA polymerase II.
  • Analysis of immunoprecipitated complexes using SDS-PAGE and radiolabeling ([35S]methionine and 32P).
  • Quantification of subunit stoichiometry and identification of phosphorylated subunits.

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Main Results:

  • Immunoprecipitation identified 10 polypeptides consistent with known RNA polymerase II subunits.
  • Relative stoichiometry of subunits was determined.
  • RPB1, RPB2, and RPB6 were identified as phosphorylated subunits.
  • Approximately 50% of RNA polymerase II molecules contained a phosphorylated RPB1 subunit.

Conclusions:

  • The study provides a more precise definition of the subunit composition of eukaryotic RNA polymerase II.
  • It elucidates the in vivo phosphorylation patterns of key subunits, particularly RPB1.
  • These findings contribute to a deeper understanding of RNA polymerase II regulation and function.