Purification of the three nuclear RNA polymerases from Neurospora crassa.
Nuclear RNA polymerases I, II, and III from Neurospora crassa have been purified 3,000-, 1,500-, and 10,000-fold, respectively, by a procedure that minimizes proteolysis of the 220-kDa subunit of polymerase II. The Neurospora enzymes resemble, in polypeptide composition, the corresponding polymerases isolated from other eukaryotes. The 220-kDa subunit of Neurospora polymerase II cross-reacts with antisera directed against the 220-kDa subunits of type II polymerases from Drosophila and wheat germ. However, the proteolyzed 180-kDa derivative of the Neurospora 220-kDa subunit fails to cross-react with the heterologous antisera, suggesting that the region removed by proteolysis contains or contributes to structural features of the enzyme that have been highly conserved during evolution. A 700-kDa complex of 12 polypeptides was found associated with polymerase II during purification. The complex was eventually separated from the enzyme, and its properties suggest that it might be associated with polymerase II in the nucleus. We describe two additional examples of polypeptides associated in variable amounts with Neurospora polymerase II.
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D000818:Animals; D005075:Biological Evolution; D002467:Cell Nucleus / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D012318:RNA Polymerase I / Q000302:isolation & purification*; D012319:RNA Polymerase II / Q000302:isolation & purification*; D012320:RNA Polymerase III / Q000302:isolation & purification*; D013045:Species Specificity
D046911:Macromolecular Substances; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D012320:RNA Polymerase III