Short CT-rich motifs can trigger context-specific silencing of gene expression in bacteria

We use an oligonucleotide library of over 10000 variants together with a synthetic biology approach to identify an insulator sequence encoded within a subset of σ54 promoters. Insulation manifests itself as silencing of expression of a downstream gene during transcriptional read through. Insulation 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 provide evidence using modeling, mutations to the CT-rich motif, and gene expression measurements on multiple sequence variants, that insulation is likely caused by binding of the RBS region to the upstream CT-rich motifs. We show that the strength of the insulation effect depends on the location and number of CU-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, indicating that there is an important regulatory role for these sequences in natural contexts. Our findings have important implications for understanding SNP/INDEL mutations in regulatory regions and add to existing guidelines for designing synthetic promoters in bacterial systems.