Final Report Summary - FESREG (Regulation of the Fe-S biogenesis in the face of stress: dynamics and involvement of new sRNAs)
Regulation of Fe-S biogenesis in the face of stress: dynamics and involvement of new sRNAs
Iron-Sulfur (Fe-S) cofactors containing proteins are key players in a plethora of biological pathways. Despite their essential roles, and because of the vulnerability of Fe/S cofactors, these proteins are not only highly prone to stress induced damage but also are internal “stress inducers” themselves as they can release free Fe2+ which produces toxic radicals by reacting with oxygen. Cells have thus designed multiple intricate ways to maintain Fe-S proteins integrity and to avoid Fe-S proteins to become the Achilles’ heel of the cell. A major stress defense mechanism is the fine control of Fe/S biogenesis under stress conditions. Remarkably, the model bacterium E. coli has the two most conserved Fe-S biogenesis systems, Isc and Suf that share biochemical features, yet differ under many structural and genetic aspects. E. coli thus constitutes an ideal model for the study of Fe-S biogenesis.
The main focus of FeSReg project has been to understand the mechanisms underlying the control of Fe-S biogenesis under stress in E. coli. The FeSReg project is articulated around two major objectives. The first is to explore if and how small regulatory RNAs (sRNAs), known to be involved in the control of many stress-responses pathways, may participate in the regulation of Fe-S biogenesis. In the last decades, sRNAs have emerged as major players in the control of gene expression in all kingdoms of life. The second objective was to better understand how genetic regulations affects Fe-S biogenesis in a dynamic manner. Indeed, despite the importance of Fe-S biogenesis in stress response, relatively little was known about when and how the cells chose in between different Fe-S biogenesis routes in function of environmental conditions.
During the course of the project, we were able to identify that one sRNA, RyhB, regulates the expression of the ensemble of Fe-S biogenesis factors. RyhB is a sRNA that is expressed under iron starvation conditions. This sRNA targets many genes by basepairing to their mRNAs and decreasing their translation and/or inducing their degradation. Likewise, RyhB reallocates iron usage in the when iron is scarce. With the FeSREg project, we were in particular able to show that RyhB represses the expression of erpA which encodes an essential Fe-S biogenesis factor during aerobic growth. In addition IscR, the major Fe-S homeostasis regulator, also represses expression of erpA but under iron rich conditions. Thus, when iron is scarce, IscR transcriptional repression is alleviated but RyhB represses expression of erpA post-transcriptionally. The combination of de-repression by IscR and repression by RyhB is best described as an incoherent circuit. These regulatory motifs are known to induce transient pulses of expression. Accordingly, expression of erpA in function of iron concentration has a typical “bell shape”: erpA expression is minimal either when iron is abundant (because of IscR repression) or when it is scarce (because of RyhB repression), but maximal at moderate iron concentrations (because both repressions are alleviated). We have found that three other major actors of Fe-S cluster biogenesis are also regulated by RyhB/IscR: nfuA, the other ATC, as well as the two major systems for Fe-S biogenesis, suf and isc (manuscript in preparation). This last operon has been shown to be regulated by IscR and RyhB, but the incoherent circuit resulting from this double regulation had not been investigated. The fact that RyhB and IscR are involved in the control of Fe-S cluster biogenesis, forming a co-regulon, was not expected.
Finally, we have already established the kinetics of expression of an actors of Fe-S biogenesis in conditions of stress. Interestingly, preliminary results show that expression varies quite rapidly when iron is chelated in the medium, but kinetics of the reaction is affected in certain sRNA mutants. While more experiments are needed to confirm these results, this already indicate that modifying the kinetics of responses to environment variations may have a more important impact than on the overall extent of the responses. Given the importance of these results in the field of RNA regulation, this part of the project will be of the highest priority to test in the very near future.
Iron-Sulfur (Fe-S) cofactors containing proteins are key players in a plethora of biological pathways. Despite their essential roles, and because of the vulnerability of Fe/S cofactors, these proteins are not only highly prone to stress induced damage but also are internal “stress inducers” themselves as they can release free Fe2+ which produces toxic radicals by reacting with oxygen. Cells have thus designed multiple intricate ways to maintain Fe-S proteins integrity and to avoid Fe-S proteins to become the Achilles’ heel of the cell. A major stress defense mechanism is the fine control of Fe/S biogenesis under stress conditions. Remarkably, the model bacterium E. coli has the two most conserved Fe-S biogenesis systems, Isc and Suf that share biochemical features, yet differ under many structural and genetic aspects. E. coli thus constitutes an ideal model for the study of Fe-S biogenesis.
The main focus of FeSReg project has been to understand the mechanisms underlying the control of Fe-S biogenesis under stress in E. coli. The FeSReg project is articulated around two major objectives. The first is to explore if and how small regulatory RNAs (sRNAs), known to be involved in the control of many stress-responses pathways, may participate in the regulation of Fe-S biogenesis. In the last decades, sRNAs have emerged as major players in the control of gene expression in all kingdoms of life. The second objective was to better understand how genetic regulations affects Fe-S biogenesis in a dynamic manner. Indeed, despite the importance of Fe-S biogenesis in stress response, relatively little was known about when and how the cells chose in between different Fe-S biogenesis routes in function of environmental conditions.
During the course of the project, we were able to identify that one sRNA, RyhB, regulates the expression of the ensemble of Fe-S biogenesis factors. RyhB is a sRNA that is expressed under iron starvation conditions. This sRNA targets many genes by basepairing to their mRNAs and decreasing their translation and/or inducing their degradation. Likewise, RyhB reallocates iron usage in the when iron is scarce. With the FeSREg project, we were in particular able to show that RyhB represses the expression of erpA which encodes an essential Fe-S biogenesis factor during aerobic growth. In addition IscR, the major Fe-S homeostasis regulator, also represses expression of erpA but under iron rich conditions. Thus, when iron is scarce, IscR transcriptional repression is alleviated but RyhB represses expression of erpA post-transcriptionally. The combination of de-repression by IscR and repression by RyhB is best described as an incoherent circuit. These regulatory motifs are known to induce transient pulses of expression. Accordingly, expression of erpA in function of iron concentration has a typical “bell shape”: erpA expression is minimal either when iron is abundant (because of IscR repression) or when it is scarce (because of RyhB repression), but maximal at moderate iron concentrations (because both repressions are alleviated). We have found that three other major actors of Fe-S cluster biogenesis are also regulated by RyhB/IscR: nfuA, the other ATC, as well as the two major systems for Fe-S biogenesis, suf and isc (manuscript in preparation). This last operon has been shown to be regulated by IscR and RyhB, but the incoherent circuit resulting from this double regulation had not been investigated. The fact that RyhB and IscR are involved in the control of Fe-S cluster biogenesis, forming a co-regulon, was not expected.
Finally, we have already established the kinetics of expression of an actors of Fe-S biogenesis in conditions of stress. Interestingly, preliminary results show that expression varies quite rapidly when iron is chelated in the medium, but kinetics of the reaction is affected in certain sRNA mutants. While more experiments are needed to confirm these results, this already indicate that modifying the kinetics of responses to environment variations may have a more important impact than on the overall extent of the responses. Given the importance of these results in the field of RNA regulation, this part of the project will be of the highest priority to test in the very near future.