Genetic context effects can override canonical cis regulatory elements in Escherichia coli

Abstract

Recent experiments have shown that in addition to control by cis regulatory elements, the local chromosomal context of a gene also has a profound impact on its transcription. Although this chromosome-position dependent expression variation has been empirically mapped at high-resolution, the underlying causes of the variation have not been elucidated. Here, we demonstrate that 1 kb of flanking, non-coding synthetic sequences with a low frequency of guanosine and cytosine (GC) can dramatically reduce reporter expression compared to neutral and high GC-content flanks in Escherichia coli. Natural and artificial genetic context can have a similarly strong effect on reporter expression, regardless of cell growth phase or medium. Despite the strong reduction in the maximal expression level from the fully-induced reporter, low GC synthetic flanks do not affect the time required to reach the maximal expression level after induction. Overall, we demonstrate key determinants of transcriptional propensity that appear to act as tunable modulators of transcription, independent of regulatory sequences such as the promoter. These findings provide insight into the regulation of naturally occurring genes and an independent control for optimizing expression of synthetic biology constructs.

Insulation of ribosomal promoter activity by Fis, H-NS or a divergent promoter within the packed E. coli genome.

“…In order to measure promoter activity independently of gene copy number effects, we normalized the promoter activity values by the gene copy number expected for that genome position at the growth rate of the sample (see Materials and Methods and ( 31)). The results obtained with the P1short promoter show that there are significant differences in promoter activity depending on insertion position (Figure 3), as observed previously for the lac and TetO1 promoters (5,32,48). We decided to compare the activity of the promoter in the presence and absence of the divergently oriented KanR cassette mainly because transcription of the Kan gene can create negative supercoiling upstream of the RNA polymerase (RNAP) and increase the expression of the rrnBP1 promoter, known for its sensitivity to the levels of negative supercoiling (58).…”

“…Previous studies have used a similar strategy in order to determine whether genome position could influence gene expression in E. coli as well as in other bacterial strains (5,31,32,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). One of the main conclusions from these studies is that the results are strongly dependent on the choice of promoter used.…”

In VivoSampling of Intracellular Heterogeneity ofPseudomonas putidaEnables Multiobjective Optimization of Genetic Devices

“…As a matter of fact, the physical location of genes in the bacterial chromosome seems not to be casual − but instead likely to reflect one more molecular stratagem for cracking otherwise intractable adaptation challenges. In sum, the data above adds to the growing evidence indicates that genetic context effects can override cis regulatory elements, pinpointing such context variability as a principal mechanism to evolutionarily optimize input–output functions in vivo .…”

Promoter capture drives the emergence of proto-genes inEscherichia coli

“…SNPs and small indels represent over 95% of mutations that we observed in the LTEE, so their relatively minor contribution to the emergence of proto-genes seems puzzling given that functional promoters are pervasive in sequence space [54,55] and occur frequently in bacterial genomes [56]. However, bacteria have several mechanisms, such as H-NS [57], transcription termination factors [58], and genetic context [59], that prevent profligate and nonproductive transcription, whereas the capture of existing promoters can bypass these requirements.…”