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...

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces ofgenetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression ofRNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.DNA topoisomerase (TOPO) I, originally known as o, is the major DNA relaxing activity in Escherichia coli (1). The gene encoding E. coli DNA TOPO I topA was localized to the cys-trp region of the chromosome (2, 3). Deletions that remove both cysB and topA were identified in E. coli strains and it was first assumed that topA was not an essential gene (3). A more careful analysis showed that a deletion of the topA gene could only be inherited in strains that contained a second mutation that would compensate for the loss of topA (4, 5). These studies identified mutations in both gyrA and gyrB that could compensate for the loss of topA and demonstrated that these mutations result in reduced global DNA supercoiling. This lead to a picture in which a "global balance" of DNA supercoiling is essential and controlled by the competing activities of DNA gyrase and TOPO I.In vitro DNA TOPO I does not relax negatively supercoiled DNA to completion (1). Other in vitro studies show that the specificity for negatively supercoiled DNA derives from a requirement of a short single-stranded DNA region as part of the enzyme-DNA complex (6). Since negative DNA supercoiling favors the unpairing of DNA strands, DNA molecules with high negative superhelical density will be a better substrate for DNA TOPO I. Some in vivo studies also suggest that DNA TOPO I does not relax DNA efficiently: in one study a topA mutation had little effect on the rate of in vivo DNA relaxation after the inhibition of DNA gyrase by coumermycin (7); and in a second in vivo study, DNA TOPO I produced only slow and partial DNA relaxation (8). However, a defect in DNA TOPO I can cause an in vivo increase in the level of negative supercoiling (5). Thus, these results suggest that DNA TOPO I functions to prevent DNA supercoiling from reaching an unacceptably high level.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3526Hypernegatively supercoiled DNA can, under certain circumstances, be produced during transcription (9, 10). In...

During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. However, we report that the 3'ETS of the glyW-cysT-leuZ polycistronic tRNA precursor is highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs are shown to base pair with the same region in the 3'ETS of leuZ (3'ETS(leuZ)). Disrupting the pairing by mutating 3'ETS(leuZ) strongly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3'ETS(leuZ) prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and decreases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3'ETS(leuZ) functions as a sponge to absorb transcriptional noise from repressed sRNAs. Additional data showing RybB and MicF sRNAs are co-purified with ITS(metZ-metW) and ITS(metW-metV) strongly suggest a wide distribution of this phenomenon.

SummaryRyhB is a small RNA (sRNA) that downregulates about 20 genes involved in iron metabolism. It is expressed under low iron conditions and pairs with specific mRNAs to trigger their rapid degradation by the RNA degradosome. In contrast to this, another study has suggested that RyhB also activates several genes by increasing their mRNA level. Among these activated genes is shiA, which encodes a permease of shikimate, an aromatic compound participating in the biosynthesis of siderophores. Here, we demonstrate in vivo and in vitro that RyhB directly pairs at the 5Ј-untranslated region (5Ј-UTR) of the shiA mRNA to disrupt an intrinsic inhibitory structure that sequesters the ribosome-binding site (ShineDalgarno) and the first translation codon. This is the first demonstration of direct gene activation by RyhB, which has been exclusively described in degradation of mRNAs. Our physiological results indicate that the transported compound of the ShiA permease, shikimate, is important under conditions of RyhB expression, that is, iron starvation. This is demonstrated by growth assays in which shikimate or the siderophore enterochelin correct the growth defect observed for a ryhB mutant in iron-limited media.

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

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