A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli - PubMed (original) (raw)

A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli

Eric Massé et al. Proc Natl Acad Sci U S A. 2002.

Abstract

A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA down-regulates 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 iron-containing proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(A) RyhB is oriented counterclockwise on the E. coli chromosome. Residues identical for 3/5 positions are shown with capital letters. The +1 site is as predicted by Argaman et al. (11) and agrees with our predictions from the alignment of the promoter. The asterisk is the start site of the lacZ fusion used in ref. . The predicted Fur sites are from ref. . The accession number for ryhB is AF480876. (B) Predicted secondary structure for ryhB from MFOLD (21).

Figure 2

Figure 2

Complementarity between the sdhCDAB operon and RyhB. Genes of the sdhCDAB operon are shown in A. Lines marked EM8 and EM9 show the position of the oligonucleotide probes used for Northern blots (Fig. 3). B shows the predicted interaction between RyhB and the sdhCDAB sense strand. The ribosome binding site for sdhD is underlined. The start codon for sdhD is shown underlined and in italics, and the stop codon for sdhC is shown in gray.

Figure 3

Figure 3

Cells were grown in LB (RyhB and sdhCDAB, lanes 1–6) or M63 glycerol (RyhB and sdhCDAB , lanes 7 and 8; and sdhC, lanes 1–6) to an OD600 0.3 and 2,2′-dipyridyl was added to a final concentration of 250 μM where indicated. A sample was removed 15′ after chelator addition; RNA was isolated as described in Materials and Methods. For lanes 1, 3, and 7, a fur+ ryhB+ strain (EM1055) was used; for lanes 2, 4, and 8, a fur+ Δ_ryhB_1∷cat strain, EM1238, was used. Cells for lanes 5 (_fur_− ryhB+) and 6 (_fur_− Δ_ryhB_1∷cat) were strains EM1256 and EM1257, respectively. [Top (RyhB)] For determination of RyhB amount, 3 μg of total RNA samples extracted from cells grown in LB (lanes 1–6) or M63 glycerol media (lanes 7 and 8) were loaded on an 8% PAGE gel. After migration, a Northern blot analysis was performed with oligoprobe EM1 (Table 1). [Middle (sdhCDAB)] Ten micrograms of the same total RNA extracts as in Top were loaded on a denaturating 1.2% agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for sdhC (probe EM8 shown in Fig. 2, Table 1). [Bottom (sdhC)] Three micrograms of RNA extracted from cells grown in M63 glycerol media were loaded on a 4% PAGE gel. A Northern blot was performed by using oligonucleotide probe EM8 within sdhC.

Figure 4

Figure 4

The same RNA samples used in lanes 1–6 of Fig. 3 were loaded on 1.2% agarose gels and probed with oligoprobes for acnA (probe EM26), fumA (probe EM23), icd (probe EM28), sodB (probe EM33), and bfr (probe EM32). FtnA expression was found to be very low in cells grown in LB, even in the absence of ryhB. Therefore, cells were grown in M63 glycerol medium and their total RNA was extracted and migrated as for the other blots, and probed with probe EM31 (ftn). Because RyhB expression is already high in this medium, induction with chelator was not carried out.

Figure 5

Figure 5

Model of Fur and RyhB interaction to regulate iron utilization.

Similar articles

Cited by

References

    1. Wassarman K M, Storz G. Cell. 2000;101:613–623. - PubMed
    1. Keiler K C, Waller P R H, Sauer R T. Science. 1996;271:990–993. - PubMed
    1. Sledjeski D D, Gupta A, Gottesman S. EMBO J. 1996;15:3993–4000. - PMC - PubMed
    1. Majdalani N, Chen S, Murrow J, St. John K, Gottesman S. Mol Microbiol. 2001;39:1382–1394. - PubMed
    1. Wassarman K M, Zhang A, Storz G. Trends Microbiol. 1999;7:37–45. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources