Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.
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.
Edwards
A M
AM
Department of Cell Biology, Stanford University School of Medicine, California 94305.
Kane
C M
CM
Young
R A
RA
Kornberg
R D
RD
D002467:Cell Nucleus / Q000378:metabolism; D002872:Chromosome Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007700:Kinetics; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012319:RNA Polymerase II