Abstract
BackgroundThere is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA–protein interactions.ResultsDNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard–Bishop–Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10−4 cannot lead to detectable variations in the DNA–protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region.ConclusionsBoth theoretical and experimental analyses of the L09 promoter’s leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.
Highlights
There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering
Inspired by the observed long-range effect of a flanking single-nucleotide polymorphism on changing the binding affinity of the eukaryotic YY1 transcription factor [9], we investigated whether a flanking point mutation would have a similar effect on the binding characteristics of the L09 promoter in comparison with the L10, L11, and L12 promoters
Trivial difference in DNA opening probability profiles between the L09 promoter and other L promoters To better understand the effect of a point mutation on the DNA opening probability profile, the 160 bp DNA fragments of the L09, L10, L11, and L12 promoters shown in Fig. 1 were re-analyzed with the Extended nonlinear Peyrard–Bishop–Dauxois (EPBD) model
Summary
There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA–protein interactions. Transcription regulation plays a major role in controlling gene expression and in altering biochemical reactions catalyzed by these gene products It has high impact in synthetic biology and metabolic. We determined the kinetics of the interaction between TetR and each promoter using a surface plasmon resonance (SPR) biosensor assay
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