Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5′–untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.
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We thank T. Yoshida for providing access to the UV spectrophotometer; E. Protozanova for discussions and advice with RNA melting experiments; I. Smolina for help and advice with reverse transcription experiments; W. Blake, J. Hasty, D.H. Lee, J. Graber and members of our lab for helpful discussions and advice in preparing the manuscript. This work was supported by the National Science Foundation and Defense Advanced Research Projects Agency.
The authors declare a pending patent application whose value may be affected by publication of this paper.
List of plasmids in this study. aThe pBADHisA vector was obtained from Invitrogen. (PDF 32 kb)
Sequences of cis-repressed RNA constructs, loop containing the YUNR (TTGG) recognition motif, ribosome binding site (RBS), and trans-activating RNA constructs used in this work. (PDF 80 kb)
Real-competitive PCR assay design. List of primers used to amplify RTPCR products obtained from RNA cell preparations. A terminator mix contains three different ddNTPs and one dNTP. For example, CGT mix for 16S rRNA is ddCTP/ddGTP/ddTTP/dATP. (PDF 9 kb)
Rational attempts to increase dynamic range of taR12-crR12 (PDF 100 kb)
Set of plasmids used in the artificial riboregulator systems. (PDF 49 kb)
Reverse transcription profiles of taRNA-crRNA complexes. (PDF 140 kb)
Determination of equilibrium dissociation constants for the taR7-crR12 pair. (PDF 34 kb)
RNA Melting curves for crR7, crR10, and crR12. Absorbance measurements at 260 nm (OD260) were determined between 10–95°C for each construct. (PDF 47 kb)
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Isaacs, F., Dwyer, D., Ding, C. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol 22, 841–847 (2004). https://doi.org/10.1038/nbt986
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