Improving the gene-regulation ability of small RNAs by scaffold engineering in Escherichia coli

Noncoding small RNAs are involved in transcriptional and post-transcriptional gene regulation of target mRNAs by modulating mRNA elongation, stability, or translational efficiency. Many natural trans-encoded small RNAs contain a scaffold that allows binding of the RNA chaperone protein Hfq for conditional gene regulation. Here, we improved the gene regulation abilities of small RNAs by directly fusing the natural Escherichia coli trans-encoded small RNA-derived scaffolds, including Hfq-binding and rho-independent transcription terminator sequences, to the 3' end of the small RNAs that mediate RNA-based gene regulation. As target small RNAs to improve their gene regulation abilities, we selected small RNAs of artificial post-transcriptional riboregulators and transcriptional attenuators. Four different small RNA scaffolds were fused to the riboregulator and attenuator-derived small RNAs. Mutations were introduced into the best small RNA scaffold to improve its gene-regulation ability further. As a result, mutations predicted to stabilize the secondary structures of the small RNA scaffolds dramatically increased its ability to regulate gene expression of both the post-transcriptional riboregulator and transcriptional attenuator systems. We believe our engineered small RNA scaffolds are applicable to other RNA regulators for improving regulatory activity, and engineered small RNA scaffolds may present a valuable strategy to regulate target gene expression strongly.