SYNERGISM IN REPLICATION AND TRANSLATION OF MESSENGER-RNA IN A CELL-FREE SYSTEM

Combination of the Qbeta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recomb inant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRN A sequence inserted into RQ135(-1) RNA, an efficient naturally occurri ng Qbeta replicase template. The enhancement is associated with a repl ication asymmetry previously described for the replication of Qbeta ph age RNA in vivo; the sense (+)-strands are produced in large excess ov er the antisense (-)-strands. This, in turn, results in increased synt hesis of the functionally active DHFR. These effects are not observed when DHFR mRNAs or RQ135(-1) RNAs are used as templates, if the transl ation system is not complete, or if it is inhibited by puromycin. The coupled replication-translation of nonviral mRNA recombinants can serv e as a useful model for studying the fundamental aspects of virus ampl ification and can be implemented for large-scale protein synthesis in vitro.