4 years ago

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters
Sarah Goldberg, Shilo Ohayon, Roni Cohen, Lior Levy, Oz Solomon, Zohar Yakhini, Orna Atar, Leon Anavy, Roee Amit, Michal Brunwasser-Meirom


We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It 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 that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein.

Publisher URL: http://www.cell.com/cell-reports/fulltext/S2211-1247(17)31353-0

DOI: 10.1016/j.celrep.2017.09.063

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