Two forms of RNA polymerase B in yeast. Proteolytic conversion in vitro of enzyme BI into BII

• Published in: European journal of biochemistry Vol. 65; no. 2; p. 543
• Main Authors: Dezélée, S, Wyers, F, Sentenac, A, Fromageot, P
• Format: Journal Article
• Language: English
• Published: England 01-06-1976
• Subjects: Amanitins; Chromatography; DNA-Directed RNA Polymerases - isolation & purification; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Isoenzymes - isolation & purification; Kinetics; Macromolecular Substances; Molecular Weight; Peptide Hydrolases; Saccharomyces cerevisiae - enzymology
• ISSN: 0014-2956
• DOI: 10.1111/j.1432-1033.1976.tb10372.x

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465 000) and 435 000, respectively). The two 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 two enzymes is in the molecular weight of the heaviest subunit which is 220 000 for enzyme BI and 180 000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 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 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 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.