Replication of RNA viruses: Structure of a 6 s RNA synthesized by the Qβ RNA polymerase

Abstract

The in vitro synthesis of RNA catalyzed by the Qβ RNA polymerase has been studied using a single-stranded 6 s RNA template. Whereas Qβ RNA replication results in the synthesis predominantly of single-stranded Qβ RNA, the predominant reaction product of 6 s RNA replication was found to be double stranded. When treated with formaldehyde to dissociate complementary base pairs, 6 s RNA exhibited a decrease in molecular weight as indicated by its slower sedimentation rate and faster electrophoretic mobility. 6 s RNA also exhibited a hyperchromic thermal transition indicative of double-stranded RNA and differing markedly from that of single-stranded RNA. The T m of this transition increased linearly with the logarithm of ionic strength. Renaturation of 6 s RNA below the T m occurred slowly and was also dependent upon ionic strength. Further analysis indicated that a small amount of RNA was in a singlestranded configuration. The frequencies of complementary dinucleotides in purified 6 s RNA were not equal and 10 to 20% of the RNA was hydrolyzed by pancreatic ribonuclease or by Neurospora crassa endonuclease. The electrophoretic mobilities of native and nuclease-treated 6 s RNA indicated that the single-stranded RNA may be present as “tails” at one or both termini of the molecule. Template activity of 6 s RNA was observed only with heat-denatured RNA and was lost upon renaturation.