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.
Different RNA species that are replicated by Q beta replicase have related secondary structures: for both plus and minus strands, "leader" stem structures were found at their 5' termini, while their 3' termini were unpaired. Parallel structures in complementary strands rather than antiparallel ones require the occurrence of wobble pairs and other imperfections in the stem regions. To test whether the leader structures are required for replication, artificial RNA sequences were synthesized by transcription from synthetic oligodeoxynucleotides with T7 RNA polymerase and assayed for their ability to be replicated by Q beta replicase. A synthetic short RNA species known to be replicated was amplified, forming a stable quasi-species; i.e., its sequence was conserved during hundreds of replication rounds. A synthetic mutant of this sequence that stabilized the leader in one strand but favored a 3'-terminal stem in the other one led to the complete loss of template activity. When new RNA sequences with the described structural requirements were designed and synthesized, their template activity was too low to be directly measurable; however, incubation with replicase produced replicating RNA whose sequence was closely related to the synthesized RNA species. The most likely interpretation is that the designed sequences were in a low montainous region in the replication fitness landscape and were optimized during amplification by Q beta replicase to a nearby fitness peak. The structural features postulated to be required for replication were not only conserved but even improved in the outgrowing mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
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