A synthetic oligo library and sequencing approach reveals an insulation mechanism encoded within bacterial σ54 promoters. Escherichia coli

We use an oligonucleotide library of over 10000 variants together with a syntheticbiology approach to identify an insulation mechanism encoded within a subset of σ54promoters. Insulation manifests itself as dramatically reduced protein expression for adownstream gene that may be expressed by transcriptional read-through. Theinsulation we observe is strongly associated with the presence of short CT-rich motifs(3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of thesilenced gene. We hypothesize that insulation is effected by binding of the RBS to theupstream CT-rich motif. We provide evidence to support this hypothesis usingmutations to the CT-rich motif and gene expression measurements on multiplesequence variants. Modelling is also consistent with this hypothesis. We show that thestrength of the silencing, effected by insulation, depends on the location and number ofCT-rich motifs encoded within the promoters. Finally, we show that in E.coli theseinsulator sequences are preferentially encoded within σ54 promoters as compared toother promoter types, suggesting a regulatory role for these sequences in naturalcontexts. Our findings suggest that context-related regulatory effects may often be dueto sequence-specific interactions encoded sparsely by short motifs that are not easilydetected by lower throughput studies. Such short sequence-specific phenomena canbe uncovered with a focused OL design that filters out the sequence noise, asexemplified herein.