Genetically Engineered Active Q[beta] Replicase in Rabbit Reticulocyte Cell-Free System

Qβ replicase functioning in Escherichia coli is an RNA-dependent RNA polymerase composed of one phage-coded subunit and three host-coded proteins: ribosomal protein S1, and protein elongation factors EF-Tu and EF-Ts. Qβ replicase lacking ribosomal protein S1 (α-less replicase) is capable of replicating some small RNAs. We attempted to create functional α-less replicase by co-expression of the mRNAs that code for the subunits of α-less replicase in a rabbit reticulocyte cell-free translation system. Replicase activity, however, could not be detected when both EF-Tu and EF-Ts were co-expressed with the phage-coded subunit. On the other hand, active α-less replicase was obtained when an EF-Ts-EF-Tu fusion protein was co-expressed with the phage-coded subunit. Consequently, we succeeded in generating genetically engineered active α-less Qβ replicase which functions in a eukaryotic cell-free system.