COUPLED REPLICATION-TRANSLATION OF AMPLIFIABLE MESSENGER-RNA - A CELL-FREE PROTEIN-SYNTHESIS SYSTEM THAT MIMICS VIRAL-INFECTION

Amplifiable messenger RNAs (Wu, Y., Zhang, D. Y., and Kramer, F. R. (1 992) Proc. Natl. Acad. Sci. U. S. A. 89, 11769-11773) were used as tem plates in coupled replication-translation reactions. These amplifiable mRNAs contained a preselected messenger sequence embedded within the sequence of MDV-1 RNA, which is a small, naturally occurring template for Qbeta replicase. When these recombinant mRNAs were incubated in vi tro in reactions that contained both an Escherichia coli cell-free tra nslation system and Qbeta replicase, the encoded protein was synthesiz ed more efficiently than in corresponding reactions that did not conta in Qbeta replicase. Moreover, when coupled replication-translation rea ctions were carried out in a continuous-flow format (Spirin, A. S., Ba ranov, V. I., Ryabova, L. A., Ovodov, S. Yu., and Alakhov, Yu. B. (198 8) Science 242, 1162-1164), the synthesis of biologically active prote in continued for a prolonged period. The results suggest that the mech anism of replication and translation in coupled reactions is similar t o the mechanism by which Qbeta phage genomic RNA is simultaneously rep licated and translated in Qbeta-infected E. coli: protein synthesis oc curs on nascent RNA strands; many more sense strands are synthesized t han antisense strands; and the integrity of the messenger sequence is preserved because a relatively small number of antisense strands serve as master templates for the synthesis of new messenger strands.