Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron–sulfur [Fe–S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe–4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe–S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.
Iron is an essential nutrient which must be provided in sufficient amounts to support growth of eukaryotic cells. All organisms devote specialized pathways to ensure proper delivery. Yet, a quantitative assessment of the intra-cellular iron concentration needed to allow the cell cycle to proceed in mammalian cells is missing. Starting from iron-depleted cell lines or primary hematopoietic progenitors prepared with clinically implemented iron chelators, replenishment via transferrin and other iron sources has been quantitatively monitored through the main endogenous markers of the cellular iron status, namely proteins involved in the uptake (transferrin receptor), the storage (ferritin), and the sensing (Iron Regulatory Proteins) of iron. When correlated with measurements of iron concentrations and indicators of growth, this minimally intrusive approach provided an unprecedented estimate of the intracellular iron concentration acting upon iron-centered regulatory pathways. The data were analyzed with the help of a previously developed theoretical treatment of cellular iron regulation. The minimal cellular iron concentration required for cell division was named functional iron concentration (FIC) to distinguish it from previous estimates of the cellular labile iron. The FIC falls in the low nanomolar range for all studied cells, including hematopoietic progenitors. These data shed new light on basic aspects of cellular iron homeostasis by demonstrating that sensing and regulation of iron occur well below the concentrations requiring storage or becoming noxious in pathological conditions. The quantitative assessment provided here is relevant for monitoring treatments of conditions in which iron provision must be controlled to avoid unwanted cellular proliferation.
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