Replication of RNA viruses. VIII. Direction of chain growth in the Qβ RNA polymerase reaction

The reaction catalyzed by the Qβ RNA polymerase isolated from Qβ phage-infected Escherichia coli has been examined with respect to the direction of RNA synthesis. Two alternative mechanisms were considered: that chain elongation is either (a) from the 5′- to the 3′-terminus with the addition of nucleotides to the 3′-hydroxyl end of the molecule, or (b) from the 3′- to the 5′-terminus, with a nucleoside triphosphate growing point. The results indicate that all RNA synthesis in this reaction is initiated by the incorporation of guanosine triphosphate at a 5′-triphosphate terminus. The radioactivity incorporated as [γ-³²P]guanosine triphosphate into growing chains of Qβ RNA or the strand complementary to Qβ RNA was not displaced when an excess of unlabeled guanosine triphosphate was added to the reaction mixture. No other ribonucleoside triphosphate, e.g. [γ-³²P]adenosine triphosphate, was incorporated. The incorporation of [γ-³²P]guanosine triphosphate into Qβ RNA upon the initiation of synthesis, and not upon chain elongation or termination, was also shown by pulse-chase techniques and zone centrifugation of the RNA product. It is therefore concluded that the direction of growth of both Qβ RNA and the strand complementary to Qβ RNA is from the 5′-triphosphate to the 3′-hydroxyl terminus. The observation that guanosine triphosphate is the 5′-terminus of the complementary strand indicates that the Qβ RNA polymerase reaction in vitro does not give rise to a complete complementary copy of Qβ RNA since the hydroxyl terminus of the phage RNA is adenosine, not cytidine.