Proteins that contain iron-sulfur (Fe-S) clusters are essential for fundamental cellular functions such as metabolism and regulation of transcription and translation as well as specialized processes like nitrogen fixation and photosynthesis. While much in known regarding individual Fe-S cluster types and the proteins that contain them, we are just beginning to unravel the complex mechanism of cluster biogenesis. One obstacle to our studies of Fe-S cluster assembly is the lack of assays to monitor cluster biogenesis in tissues and living systems in high throughput. Here, we describe fluorescent bisosensors that monitor cluster biogenesis in complex samples and mammalian cell culture. The described sensors allow for kinetic analysis of Fe-S cluster biogenesis and decay in complex mixtures, function in both the mitochondria and the cytosol and are sensitive to changes in cellular state. The described biosensors should enable clinical evaluation of diseased tissues and allow for high-throughput screens for genes and small molecule effectors of cluster biogenesis.

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