A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro.
The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.
D000645:Ammonium Sulfate / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005796:Genes; D009154:Mutation; D010455:Peptides / Q000032:analysis; D012319:RNA Polymerase II / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D010455:Peptides; D012331:RNA, Fungal; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D000645:Ammonium Sulfate