Novel Vector for Generating RNAs with Defined 3′ Ends and Its Use in Antiviral Strategies

Abstract

A novel transcription system was constructed that allows trimming of 3′ termini of RNA transcripts in E. coli by endogenous RNase P. Here, the sequence of tRNASer from E. coli fused downstream of the target sequence directs posttranscriptional cleavage 3′ of the target sequence. As a first-target MNV11(+), a self-replicating RNA from the Qβ system was subjected to transcription in vivo. Northern blotting experiments revealed that the primary transcript was indeed successfully processed to an RNA of expected length. The RNA released proved to function as an active template for Qβ replicase. Moreover, E. coli cells producing these short-chain replicator molecules no longer supported multiplication of Qβ phages upon infection. Since the novel transcript-trimming system utilizes the endogenous RNase P activity and does not depend on any particular 3′-terminal RNA sequence of target molecules, it may have wide applications for a number of different targets in prokaryotes. Further applications, including those in eukaryotes, are discussed.