title: RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties.
abstract: An RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Schiebel
authors.forename: B
authors.initials: B
authors.lastname: Haas
authors.forename: S
authors.initials: S
authors.lastname: Marinković
authors.forename: A
authors.initials: A
authors.lastname: Klanner
authors.forename: H L
authors.initials: HL
authors.lastname: Sänger
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010942:Plant Viruses / Q000502:physiology; D010944:Plants / Q000201:enzymology* / Q000382:microbiology; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D012313:RNA; D012324:RNA-Dependent RNA Polymerase