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.
| Original language | English (US) |
|---|---|
| Article number | e1007405 |
| Journal | PLoS genetics |
| Volume | 14 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2018 |
| Externally published | Yes |
Bibliographical note
Funding Information:The authors acknowledge financial support for this work from the NIH (www.nih.gov) (DP2-GM105444 and R01-GM127593 to RMK, R01-GM080279 to MG, training support T32-AI060516 for JMK, T32-GM7229 for CYM, T32-AR007442 and K08-AI127933 for MJC), the Burroughs Wellcome (www.bwfund.org) PATH Award (to RMK), and the NSF (www.nsf.gov) (CHE-1337449 to RMK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We acknowledge Zachary Hostetler for his assistance and advice with the KinTek Explorer software and preparation of the manuscript and Manuela Roggiani for guidance with strain construction.
Publisher Copyright:
© 2018 Culyba et al. http://creativecommons.org/licenses/by/4.0/