Engineering Hfq binding sequence to control gene regulation of bacterial small RNA

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Yuta Sakai, Koichi Abe, Saki Nakashima, Masataka Araki, Koji Sode, Kazunori Ikebukuro

Tokyo University of Agriculture and Technology and Japan Science and Technology Agency, Japan

Non-coding small RNAs (sRNAs) are involved in post-transcriptional gene regulation and controls cellular processes. Until now, many natural sRNAs has been identified and characterized in Escherichia coli. These sRNAs base-pair with the 5-untranslated region and/or the translation initiation region of the mRNA, thereby modulating the mRNA stability or the translational efficiency resulting in the activation or repression of the gene expression. The trans-encoded sRNAs, which are encoded separately from the mRNA, commonly binds to Hfq, an RNA chaperone protein. Hfq binds to a single-stranded AU-rich sequence of sRNA that is not within the antisense region, which hybridizes with the mRNA. This Hfq-binding promotes sRNA-mRNA hybridization and also enhances the stability by protecting sRNAs from ribonuclease degradation. Since artificial sRNAs can be designed based on the complementary sequence of the target mRNA, sRNAs are valuable to control the expression of various target genes. In this research, we engineered the Hfq binding sequence based on natural sRNAs to further improve their gene regulation ability. We selected four different E. coli-derived, Hfq-dependent sRNAs and focused on their Hfq binding sequence. The engineered Hfq binding sequences were designed by stabilizing the secondary structure and/or by introducing sequence that has high affinity against Hfq. We introduced these engineered Hfq binding sequence into the natural sRNA by substituting its natural Hfq binding sequence with the engineered one or directly fused the engineered Hfq binding sequence to the antisense sequence of the artificial sRNA. As a result, we were able to improve the gene regulation of both natural and artificial sRNAs in E. coli. We believe our engineered Hfq binding sequence would be applicable to other sRNAs to improve its regulatory activity and engineering Hfq binding sequence would be a valuable strategy to engineer sRNAs that strongly regulate the expression of their target gene.