Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter’s activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli , approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of ‘early’, ‘middle’, and ‘late’ genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene’s transcription as a function of stimulus dose. Author summary As the precise timing of gene expression is critical for cells to respond and adapt to new environments, it is important to understand the underlying mechanisms which control this timing. In this report, we studied the timing of transcription for genes in the bacterial DNA damage repair pathway (known as the SOS response), a regulatory system where each gene is controlled by the same transcriptional repressor, LexA. By specifically isolating the role of the LexA binding interaction at SOS gene promoters, we found a relationship between the amount of DNA damage incurred by the cell, LexA binding kinetics at a promoter, and the timing of promoter activation. Our data fit a kinetic model that reveals how a disequilibrium between the LexA-operator binding reaction and cellular LexA concentrations causes timing differences between genes to emerge only at higher doses of DNA damage. Taken together, we show that non-equilibrium DNA binding kinetics is the mechanism by which a single transcription factor can modulate timing differences across an entire network of genes as a function of stimulus dose.
【초록키워드】 Gene Expression, Transcription, DNA damage, activity, Regulatory, Physiology, low dose, pathway, network, Degradation, expression, Critical, mechanism, binding kinetics, binding, cellular, Concentration, dose, regulate, Trigger, Escherichia coli, kinetic, triggers, leads, transcription factor, the cell, best, Activation, DNA binding, These data, promoters, drop, operator, SOS response, promoter, Biochemical pathways, disequilibrium, DNA damage repair pathway, LexA, operators, rapid equilibrium assumptions, SOS gene, SOS pathway, transcriptional repressor, bacterial DNA, Genes, Cell, binding interaction, defined, resulting, modulate, cause, regulated, reveal, subset, respond, separated, DNA damaging, the timing, transcription regulation, 【제목키워드】 binding kinetics, dose, transcriptional,