Abstract
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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Acknowledgements
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.
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Supplementary information
Supplementary Table 1
List of plasmids in this study. aThe pBADHisA vector was obtained from Invitrogen. (PDF 32 kb)
Supplementary Table 2
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)
Supplementary Table 3
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)
Supplementary Notes
Rational attempts to increase dynamic range of taR12-crR12 (PDF 100 kb)
Supplementary Fig. 1
Set of plasmids used in the artificial riboregulator systems. (PDF 49 kb)
Supplementary Fig. 2
Reverse transcription profiles of taRNA-crRNA complexes. (PDF 140 kb)
Supplementary Fig. 3
Determination of equilibrium dissociation constants for the taR7-crR12 pair. (PDF 34 kb)
Supplementary Fig. 4
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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DOI: https://doi.org/10.1038/nbt986
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