Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis.
DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 microM for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of alpha-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from C. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of non-specific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.
Patturajan
M
M
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
D002175:Candida / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D008970:Molecular Weight; D012319:RNA Polymerase II / Q000302:isolation & purification*
D012319:RNA Polymerase II