Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli.
A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. The poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity as well as RNA polymerase activity. In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates. In the absence of added primer, RNA products up to twice the length of the template are synthesized. When incubated in the presence of a single nucleoside triphosphate, [alpha-32P]UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA. These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro.
Plotch
S J
SJ
Molecular Biology Research Section, American Cyanamid Company, Lederle Laboratories, Pearl River, New York 10965.
Palant
O
O
Gluzman
Y
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011233:Precipitin Tests; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D013698:Templates, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases