Abstract
RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.
Highlights
The RNA coliphage MS2 is one of the oldest model systems of modern molecular biology and its detailed investigation has led to numerous fundamental findings and applications
We set out to probe if the expressed rep  subunit can form an active replicase through complex formation with the proposed E. coli host factors, which are present in the PURE system [34]
We expected that the responsible RNA domains are contained in the two untranslated regions (UTRs) of the MS2 genome, each of which folds into a defined secondary structure [45]
Summary
The RNA coliphage MS2 is one of the oldest model systems of modern molecular biology and its detailed investigation has led to numerous fundamental findings and applications. Further studies led to the discovery of RNA– RNA and RNA–protein interactions that control the precise timing and strength of viral protein expression during the bacteriophage life cycle [2,3,4]. From these interactions, binding of the coat protein to a ‘translational operator’ stem–loop containing the start codon of the rep  subunit [4] has become a versatile tool in molecular and cell biology applications such as RNA imaging [5,6,7]. Both ␥ and ␦ subunit appear to act as chaperones for the rep  subunit [16] and are essential for processive RNA elongation [17,18], while the ␣ subunit seems to be necessary for RNA initiation and termination [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
