Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro

J Biol Chem. 1991 Jan 5;266(1):71-5.

Abstract

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Nucleus / metabolism
  • Chromosome Deletion
  • Genes, Fungal*
  • Genetic Complementation Test
  • Kinetics
  • Macromolecular Substances
  • Promoter Regions, Genetic*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / isolation & purification
  • RNA Polymerase II / metabolism*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Transcription, Genetic*

Substances

  • Macromolecular Substances
  • RNA Polymerase II