The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3.
A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.
Osman
T A
TA
Department of Biology, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Buck
K W
KW
D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000302:isolation & purification; D039621:Eukaryotic Initiation Factor-3; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000032:analysis*; D014027:Tobacco Mosaic Virus / Q000201:enzymology*
D039621:Eukaryotic Initiation Factor-3; D010448:Peptide Initiation Factors; D010940:Plant Proteins; D016601:RNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases