With the aim of investigating the flexibility one has when engineering a sRNA regulator based on our system we performed a bioinformatic study to elucidate sequence and structure conservation among 24 bacterial species representative of the diversity within Enterobacteriaceae.

First the ChiP homolog including its ribosome binding site (RBS) was indentified in the 24 species by BlastP using the E.Coli protein as bait. Then, to identify putative ChiXs a local BlastN search was performed with the sequence flanking the RBS as query and the given genome as target (window size of 7). Hits in coding genes were excluded since we restrict ChiX-homologs to be in intergenic regions as is the case in E.Coli and Salmonella [Overgaard et al., 2009, Figueroa-Bossi et al., 2009]. In addition, hits which did not seem to have a putative -35 and -10 in a reasonable distance from the putative start-site were also exluded, leaving one hit for each of the 24 sequences. Next, the secondary structure of the 24 putative ChiX homologs were analysed by RNAfold (default parameter setting) from the Vienna RNA package version 2.0.0. and sequences length were selected for further analysis so they start with 1 or 2 nucleotides upstream of the first stemloop and end with 4 or 5 Ts downstream of the second stem-loop. In the next step, a structural alignment was made by the LocARNA server, also from the Vienna RNA package version 2.0.0 (using default parameter settings), and finally, the RNA secondary structure and sequence conservation was visualized by RNAlogo webserver. Afterwards the percentage of G/C and A/T (out of 24) was manually calculated. Note that at gap positions these two numbers do not sum to 100.

From the sequence logo it can be seen that the structure but not the sequence is conserved in the first stemloop, whereas both the structure and sequence is conserved in the second stemloop. Another interesting feature is the conserved A/U stretch around position 40 which resemble the characteristic Hfq binding motif. The sequence which base-pair with the ChiP RBS is perfectly conserved, this might be due to evolutionary selection for a strong RBS rather than functionally constraints (AAAGAGG is not a bad RBS, and is somewhat similar to the RBS BioBricks: BBa_B0030, BBa_B0034, BBa_B0035, and BBa_B0064 which has a relative strength of 0.35-1.124). Consequently we expect that complementary mutations can freely be made to match the RBS in any mRNA of interest.

References

Figueroa-Bossi, Nara, Martina Valentini, Laurette Malleret, and Lionello Bossi. “Caught at its own game: regulatory small RNA inactivated by an inducible transcript mimicking its target.” Genes & Development 23, no. 17 (2009): 2004 -2015. http://genesdev.cshlp.org/content/23/17/2004.abstract.

Overgaard, Martin, Jesper Johansen, Jakob Møller‐Jensen, and Poul Valentin‐Hansen. “Switching off small RNA regulation with trap‐mRNA.” Molecular Microbiology 73, no. 5 (September 2009): 790-800. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06807.x/abstract.