DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity.
Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.
D000546:Amanitins / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D008670:Metals / Q000494:pharmacology; D009994:Osmolar Concentration; D010944:Plants / Q000201:enzymology; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D012320:RNA Polymerase III / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic
D000546:Amanitins; D008670:Metals; D011089:Polydeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III