A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA downregulates a set of iron-storage and iron-using proteins when iron is limiting; it is itself negatively regulated by the ferric uptake repressor protein, Fur (Ferric uptake regulator). RyhB RNA levels are inversely correlated with mRNA levels for the sdhCDAB operon, encoding succinate dehydrogenase, as well as five other genes previously shown to be positively regulated by Fur by an unknown mechanism. These include two other genes encoding enzymes in the tricarboxylic acid cycle, acnA and fumA, two ferritin genes, ftnA and bfr, and a gene for superoxide dismutase, sodB. Fur positive regulation of all these genes is fully reversed in an ryhB mutant. Our results explain the previously observed inability of fur mutants to grow on succinate. RyhB requires the RNA-binding protein, Hfq, for activity. Sequences within RyhB are complementary to regions within each of the target genes, suggesting that RyhB acts as an antisense RNA. In sdhCDAB, the complementary region is at the end of the first gene of the sdhCDAB operon; full-length sdhCDAB message disappears and a truncated message, equivalent in size to the region upstream of the complementarity, is detected when RyhB is expressed. RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential ironcontaining proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.Fur ͉ Hfq ͉ posttranscriptional regulation

RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli. When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded. RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues. We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs. Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants. RyhB requires the RNA chaperone Hfq. In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E. Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E. Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing. Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

RyhB is a noncoding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single noncoding RNA on iron homeostasis.Iron (Fe) is an essential element for virtually all organisms. This metal is an integral part of heme and is used as a cofactor in Fe-S proteins involved in major biological processes such as electron transport, the trichloroacetic acid (TCA) cycle, photosynthesis, N 2 fixation, gene regulation, and DNA biosynthesis (2). Iron is one of the most abundant elements on earth; it is readily soluble under anaerobic conditions but becomes extremely insoluble in the presence of oxygen at neutral pH. Although it is essential for the physiology of most organisms, under aerobic conditions free iron is extremely toxic because of its ability to catalyze the formation of reactive oxygen species that can damage a variety of cellular components. To overcome iron toxicity, bacteria like Escherichia coli strictly regulate iron uptake and storage according to the availability of iron in the environment.Iron can be transported actively from the environment to the cytoplasm via specific iron-binding transporters located in the bacterial membrane. Under conditions of iron limitation, E. coli cells produce the iron-chelating siderophore enterobactin (or enterochelin) (34), as well as cell surface iron transport proteins that recognize iron-loaded siderophores or other sources of iron, such as ferric citrate (12,6,13). While binding to surface proteins is energy independent, the transport through the outer membrane of iron-siderophore complexes is driven by energy-transducing proteins TonB-ExbB-ExbD, also called the TonB complex (26,23). Once inside the cell, the metal is deposited into Fe-S proteins, heme, or iron-storage proteins. Iron-storage proteins called ferritin, encoded by ftnA, and bacterioferritin, encoded by bfr, store excess intracellular iron that is not required for incorporation into cellular enzymes.When intracellular iron levels become sufficiently high, expression of the iron uptake genes is represse...