In vitro recombination and terminal elongation of RNA by Q beta replicase

Abstract

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.