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Engineered riboregulators enable post-transcriptional control of gene expression

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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Figure 1: The artificial riboregulator system used to control post-transcriptional gene regulation.
Figure 2: Results of cis repression of crRNA variants: crRL (red), crR7 (orange), crR10 (green), crRB (light blue) and control (dark blue).
Figure 3: Trans-activation mechanism and results.
Figure 4: Transient and steady-state responses and specificity results of riboregulator systems.

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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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Correspondence to James J Collins.

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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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