The bacterial promoter spacer modulates promoter strength and timing by length, TG-motifs and DNA supercoiling sensitivity

Abstract

Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.

The Role of Replication-induced Chromosomal Copy Numbers in Spatio-temporal Gene Regulation and Evolutionary Chromosome Plasticity

“…To determine the influence of promoter regulation and the copy number effect on the gene expression pattern of exponential phase compared to stationary phase, altering only one of those factors is necessary. As changes in the genetic regulatory network would be difficult due to the diversity of regulatory proteins and DNA topology 9,31,32 , we decided to significantly alter the copy number effect. By relocation of the oriC into the terminus region, we would get an opposite copy number profile compared to wild type.…”

Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria

“…Further model improvements will require taking into account additional long-distance and non-additive transcriptional interactions, for example, interference between colliding RNAPs [51][52][53] and sequencedependent sensitivities to DNA supercoiling 54,55 , as well as additional gene expression processes that ultimately affect measurements, such as changes in DNA copy numbers, transcriptional regulation, translation rates, mRNA decay rates, and coupling between these processes 5,28,56,57 . A key necessity will be to ensure both model accuracy and model generality by evaluating predictions across many datasets covering the full range of potential interactions.…”

Genetic context effects can override canonicalcisregulatory elements inEscherichia coli

“…We note that in contrast to the majority of recent work on the effects of sequence contexts on transcription, which focus on the regulatory sequences immediately upstream of a gene (the promoter region and region 100-200 bp upstream of the transcription start site), here we have kept a fixed sequence in the window 162 bp upstream of the transcription start site (and 852 bp downstream of the end of our reporter ORF), and instead focused on the longer-range impacts of genetic context. Our data demonstrate that, in parallel to the extensively studied fundamental effects of regulatory sequences (6,(34)(35)(36)(37)(38) , genetic context further than 162 bp from the transcription start site has a profound effect on transcription level, and must be considered both in efforts to model transcription of natural genes, and in the design of synthetic biology constructs.…”