Sequence analysis of RNA species synthesized by Q beta replicase without template.

Abstract

Q beta replicase amplifies certain short-chained RNA templates autocatalytically with high efficiency. In the absence of extraneously added template, synthesis of new RNA species by Q beta replicase is observed under conditions of high enzyme and substrate concentrations and after long lag times. Even under identical conditions, different RNA species are produced in different experiments. The sequences of several independent template-free products have been determined by cloning their cDNAs into plasmids by a novel cloning procedure. Their nucleotide chain lengths are small, ranging from 25 to about 50 nucleotides. While their primary sequences are unrelated except for the invariant 5'-terminal G and 3'-terminal C clusters, their tentative secondary structures show a common principle: both their plus and minus strands have a stem at the 5' terminus, while the 3' terminus is unpaired. Direct accumulation of sufficient quantities of early template-free synthesis products by Q beta replicase is prevented by the inherent irreproducibility of the synthesis process and by the rapid change of the products during amplification by evolution processes, but large amounts of such RNA can be synthesized in vitro by transcription from the cDNA clones. RNA species produced in template-free reactions replicate much more slowly than the optimized RNA species characterized previously. These experimental results illustrate how biological information can be gained in small bits by trial and error.