Two Forms of RNA Polymerase B in Yeast

Abstract

Special care to prevent proteolysis during yeast [Saccharomyces cerevisiae] RNA polymerase B purification leads to the appearance of 2 forms of enzymes, BI and BII, with different MW (465,000 and 435,000, respectively). The 2 forms of enzyme can be separated by ion-exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the 2 enzymes is in the MW of the heaviest subunit which is 220,000 for enzyme BI and 180,000 for enzyme BII. Otherwise, the 2 enzymes have 7 common subunits of MW 150,000, 45,000, 26,000, 22,500, 14,500, 12,500 and 9000. Two additional polypeptide chains of 32,000 and 16,500 Mr [relative MW] are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAE Sephadex chromatography. The largest subunit of enzyme BI (Mr 220,000) can be specifically cleaved in vitro by a yeast protease [EC 3.4.21.4] extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single-stranded or double-stranded DNA as template. The precursor-product relationship of the different forms of class B RNA polymerases in eukaryotic cells is discussed.