Does Qβ replicase synthesize RNA in the absence of template?

Abstract

Qβ replicase, in the absence of added template, will synthesize RNA autocatalytically¹. A variety of small RNA species, termed ‘6S RNAs’ are generated². As this reaction purportedly occurs in the absence of template, it has been termed ‘de novo’ RNA synthesis³. The question of whether Qβ replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Qβ replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Qβ replicase preparations³; (2) differences in the sizes of products of apparently identical reactions⁴; and (3) kinetic differences between template-instructed and de novo reactions⁵. Here we describe a procedure for production of Qβ replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Qβ replicase preparations. Thus our findings suggest that, in the de novo reaction, Qβ replicase is replicating previously undetected contaminating RNA molecules.