Abstract
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.
Highlights
Transcription regulation networks enable a cell to exert exquisite control over its biochemical pathways and are prevalent across the tree of life [1]
We studied the timing of transcription for genes in the bacterial DNA damage repair pathway, a regulatory system where each gene is controlled by the same transcriptional repressor, LexA
By 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
Summary
Transcription regulation networks enable a cell to exert exquisite control over its biochemical pathways and are prevalent across the tree of life [1]. The gene networks encoding sets of amino acid biosynthetic enzymes have been shown to be transcribed in the same order they are needed in their biochemical pathway. This phenomenon, termed just-in-time transcription, likely ensures that energy is not expended on expressing genes before their products can be fully actualized in the pathway [3]. Another remarkable aspect of the SOS pathway is that, unlike other pathways that exhibit temporal ordering of gene transcription [2, 7], the temporal ordering of SOS genes is not attributable to a serial cascade of transcription factors, but instead appears to involve only one repressor protein
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