A synthetic oligo library and sequencing approach reveals an insulation mechanism encoded within bacterial σ⁵⁴promoters

Abstract

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 σ 54 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 CTrich 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 σ 54 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.

Synthetic 5’ UTRs can either up- or down-regulate expression upon RBP binding

“…Synthetic biology approaches have been increasingly used in recent years to map potential regulatory mechanisms of transcriptional and translational regulation, in both eukaryotic and bacterial cells (56)(57)(58)(59)(60)(61). Here, we built on the design introduced by (12) to quantitatively study RBP-based regulation in bacterial 5' UTRs, using the combined synthetic biology and SHAPE-Seq approach that we recently applied to the study of hairpins with bacterial gene-header regions (40).…”

“…The first corresponds to the binding site (-56 to -30) as expected, while the second corresponds to a downstream satellite structure (-23 to -10). The secondary hairpin encodes a putative short anti-Shine-Dalgarno (aSD) motif (CUCUU) (56), which may partially sequester the RBS. While RBS-sequestration by an aSD motif can explain the up-regulation effect observed for PP7-wt, it cannot at the same time explain the down-regulatory phenomenon observed for PP7-USs, nor its high basal production rate levels.…”