(596ac) Tunable Synthetic Divergent Promoters

In bacteria, many gene pairs are transcribed in opposite directions from divergent promoters. The promoters are characterized as having closely spaced and/or overlapping binding sites for RNA polymerase. In E. coli, divergent promoters are extremely common and actually outnumber standard, unidirectional promoters. Of these divergent promoters, roughly half have overlapping RNA polymerase binding sites.

In previous work we found that divergent promoters enable novel control in gene regulation by coupling the transcription of the regulator and structural genes. In this work, we investigated the ingredients involved in engineering divergent promoters. To do this, we constructed three libraries of overlapping divergent promoters based on a previously developed synthetic promoter library for unidirectional promoters.  Clones with varying promoter strengths in both directions were obtained and analyzed to uncover the factors determining the behavior of overlapping divergent promoters.

•  Three libraries of divergent promoters were constructed using degenerate oligonucleotide sequences with consensus sequences for E. coli promoters. All show a wide range of promoter strength in both directions.

•  Ten promoters have mutations in the -10 or -35 consequence sequence. However, these mutations are not necessarily linked to reduced promoter strength.

•  Phylogenetic analysis can qualitatively indicate the importance of certain positions. However, we did not observe a strong correlation based on the phylogenetic tree.

•  Further analysis is needed to uncover the factors involved in varying promoter strengths.