Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase.
In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs. In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA. This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function. Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein. The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase. By employing an E. coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred. This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.
Mills
D R
DR
Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011777:Q beta Replicase / Q000235:genetics*; D012316:RNA Nucleotidyltransferases / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D011995:Recombination, Genetic; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases