RT Journal Article SR Electronic T1 A synthetic oligo library and sequencing approach reveals an insulation mechanism encoded within bacterial σ54 promoters JF bioRxiv FD Cold Spring Harbor Laboratory SP 086108 DO 10.1101/086108 A1 Lior Levy A1 Leon Anavy A1 Oz Solomon A1 Roni Cohen A1 Michal Brunwasser-Meirom A1 Shilo Ohayon A1 Orna Atar A1 Sarah Goldberg A1 Zohar Yakhini A1 Roee Amit YR 2017 UL http://biorxiv.org/content/early/2017/06/13/086108.abstract AB We use an oligonucleotide library of over 10000 variants together with a synthetic biology approach to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as dramatically reduced protein expression for a downstream gene that may be expressed by transcriptional read-through. The insulation 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 the silenced gene. We hypothesize that insulation is effected by binding of the RBS to the upstream CT-rich motif. We provide evidence to support this hypothesis using mutations to the CT-rich motif and gene expression measurements on multiple sequence variants. Modelling is also consistent with this hypothesis. We show that the strength of the silencing, effected by insulation, depends on the location and number of CT-rich motifs encoded within the promoters. Finally, we show that in E.coli these insulator sequences are preferentially encoded within σ54 promoters as compared to other promoter types, suggesting a regulatory role for these sequences in natural contexts. Our findings suggest that context-related regulatory effects may often be due to sequence-specific interactions encoded sparsely by short motifs that are not easily detected by lower throughput studies. Such short sequence-specific phenomena can be uncovered with a focused OL design that filters out the sequence noise, as exemplified herein.