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.

Summary: RyhB 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 5′‐untranslated region (5′‐UTR) of the shiA mRNA to disrupt an intrinsic inhibitory structure that sequesters the ribosome‐binding site (Shine‐Dalgarno) 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. Bacterial genetic regulation is mostly performed at the levels of transcription and translation. Recently discovered riboswitches are RNA molecules located in untranslated regions of messenger RNAs that modulate the expression of genes involved in the transport and metabolism of small metabolites. Several riboswitches have recently been shown to employ various regulation mechanisms, but no general rules have yet been deduced from these studies. Here, we have analyzed two adenine-sensing riboswitches of Bacillus subtilis and Vibrio vulnificus that differ by the level at which they control gene expression, which is transcription and translation, respectively. We find that, beyond the obvious difference in transcriptional and translational modulation, riboswitch regulation mechanisms of both adenine riboswitches are fundamentally different. For instance, while the adenine riboswitch from B. subtilis performs co-transcriptional binding for gene regulation, the riboswitch from V. vulnificus relies on reversible ligand binding to achieve gene regulation during mRNA translation. In agreement with co-transcriptional binding of the B. subtilis riboswitch, we find that transcriptional pausing is crucial for gene 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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