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• W2078797908 abstract "A small noncoding bacterial ribonucleic acid of 62–64 nucleotides, RydC, was identified in the genomes of Escherichia coli, Salmonella, and Shigella. In vivo, RydC binds to the RNA-binding protein Hfq, and it is unstable when Hfq is absent. Mobility assays reveal that complex formation between RydC and Hfq is specific, with an apparent binding constant of ∼300 nm. Sequence alignments combined with structural probing demonstrate that RydC folds as a pseudoknot. Hfq binds the loops crossing the deep and shallow grooves of the pseudoknotted RNA and reorganizes its overall conformation. An interaction with a polycistronic mRNA, yejABEF, which encodes a putative ABC transporter, was detected by affinity purification of immobilized RNA-Hfq complexes. In vivo, the yejABEF operon is expressed on minimal medium. Remarkably, its expression is reduced when RydC is absent, and the operon is degraded when RydC expression is stimulated. This observation correlates with the growth defects associated with a stimulation of its expression in vivo, generating a thermosensitive phenotype that affects growth on minimal media supplemented with glycerol, maltose, or ribose. We conclude that RydC regulates the yejABEF-encoded ABC permease at the mRNA level. This small RNA may contribute to optimal adaptation of some Enterobacteria to environmental conditions. A small noncoding bacterial ribonucleic acid of 62–64 nucleotides, RydC, was identified in the genomes of Escherichia coli, Salmonella, and Shigella. In vivo, RydC binds to the RNA-binding protein Hfq, and it is unstable when Hfq is absent. Mobility assays reveal that complex formation between RydC and Hfq is specific, with an apparent binding constant of ∼300 nm. Sequence alignments combined with structural probing demonstrate that RydC folds as a pseudoknot. Hfq binds the loops crossing the deep and shallow grooves of the pseudoknotted RNA and reorganizes its overall conformation. An interaction with a polycistronic mRNA, yejABEF, which encodes a putative ABC transporter, was detected by affinity purification of immobilized RNA-Hfq complexes. In vivo, the yejABEF operon is expressed on minimal medium. Remarkably, its expression is reduced when RydC is absent, and the operon is degraded when RydC expression is stimulated. This observation correlates with the growth defects associated with a stimulation of its expression in vivo, generating a thermosensitive phenotype that affects growth on minimal media supplemented with glycerol, maltose, or ribose. We conclude that RydC regulates the yejABEF-encoded ABC permease at the mRNA level. This small RNA may contribute to optimal adaptation of some Enterobacteria to environmental conditions. A number of 40–400-nt 1The abbreviations used are: nt, nucleotide(s); sRNA, small RNA; MOPS, 4-morpholinepropanesulfonic acid; RACE, rapid amplification of cDNA ends; FPLC, fast protein liquid chromatography; pCp, cytidine-3′,5′-biphosphate.1The abbreviations used are: nt, nucleotide(s); sRNA, small RNA; MOPS, 4-morpholinepropanesulfonic acid; RACE, rapid amplification of cDNA ends; FPLC, fast protein liquid chromatography; pCp, cytidine-3′,5′-biphosphate. RNAs that generally do not encode proteins or function as transfer or ribosomal RNAs have been characterized in Escherichia coli. Because of their small sizes, they have been referred to as small (s) or noncoding (nc) RNAs. Initially, a dozen sRNAs were identified in E. coli on the basis of their high abundance or by serendipity. In the last few years, computational, microarray and cloning-based screens have led to the identification of around 50 additional sRNAs in E. coli (for recent reviews, see Refs. 1Masse E. Majdalani N. Gottesman S. Curr. Opin. Microbiol. 2003; 6: 120-124Crossref PubMed Scopus (121) Google Scholar and 2Hershberg R. Altuvia S. Margalit H. Nucleic Acids Res. 2003; 31: 1813-1820Crossref PubMed Scopus (214) Google Scholar). These RNAs act mainly by pairing with other RNAs, are part of RNA-protein complexes, or adopt the structures of other nucleic acids (3Storz G. Opdyke J.A. Zhang A. Curr. Opin. Microbiol. 2004; 7: 140-144Crossref PubMed Scopus (284) Google Scholar).Bacterial sRNAs base pairing with target mRNAs can have various regulatory fates: sRNAs can repress or activate translation by blocking or promoting ribosome binding to mRNAs (3Storz G. Opdyke J.A. Zhang A. Curr. Opin. Microbiol. 2004; 7: 140-144Crossref PubMed Scopus (284) Google Scholar). They can also destabilize or stabilize mRNAs by increasing or decreasing accessibility to RNases. Base pairing between some sRNAs and their RNA targets requires the participation of the Hfq protein, a homolog of the Sm and Sm-like eukaryotic proteins involved in mRNA splicing. Hfq binds AU-rich sequences and forms a homohexameric ring (4Schumacher M.A. Pearson R.F. Moller T. Valentin-Hansen P. Brennan R.G. EMBO J. 2002; 21: 3546-3556Crossref PubMed Scopus (332) Google Scholar). It has been proposed that Hfq acts as an RNA chaperone to promote base pairing interactions between Hfq-binding sRNAs and their targets. It can protect many sRNAs and also mRNAs against RNase E digestion (5Moll I. Afonyushkin T. Vytvytska O. Kaberdin V.R. Blasi U. RNA (N. Y.). 2003; 9: 1308-1314Crossref PubMed Scopus (208) Google Scholar). The mechanisms by which Hfq facilitates interactions between sRNAs and their targets are, however, poorly understood.In this report, the functional and structural identification of a novel sRNA that folds as a pseudoknot and binds Hfq in vitro and in vivo is described. The RNA was detected in the enterobacteriaceae family, in 21 sequenced strains from the three genera Escherichia, Salmonella, and Shigella. Its existence was independently detected by microarrays of Hfq-immunoprecipitated E. coli RNAs and Northern analysis (6Zhang A. Wassarman K.M. Rosenow C. Tjaden B.C. Storz G. Gottesman S. Mol. Microbiol. 2003; 50: 1111-1124Crossref PubMed Scopus (429) Google Scholar), and it was termed RydC. We have identified by affinity chromatography an mRNA target of RydC that specifies a predicted ABC transport system. In vivo, RydC regulates the expression of the ABC permease at the mRNA level. When the expression of RydC is stimulated in vivo, the mRNA encoding the transporter gets degraded, and growth defects are observed on minimal medium with glycerol, maltose, or ribose as the carbon sources.EXPERIMENTAL PROCEDURESrydC Gene Disruption and Overexpression—The chromosomal rydC gene was deleted by targeted gene substitution using a combination of two described protocols. The cat gene flanked by FLP recognition target from pKD3 was amplified by PCR using P1 (5′-CTACGCATGATGCCGCGTAAACGTTCCTGAAAGGATATTTAGTGTAGGCTGGAGCTGCTTC-3′) and P2 (5′-GATTAAAAATAAGCCAGATGGACGTATGGGCAAGGATTATGCATATGAATATCCTCCTTAGT-3′), as described previously (7Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10970) Google Scholar). Strain KY330 was transformed with the PCR product as described previously (8Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1353) Google Scholar). Homologous recombination between the PCR product and the chromosome leads to chloramphenicol-resistant clones in which the chromosomal rydC gene is replaced by the pKD3-encoded cat gene, as confirmed by PCR using P3 (5′-CGGAATTCCGGCATCGGCATAAAAGG-3′) and P4 (5′-CGGGATCCTCTGGAAGGACAA CACAC-3′), and the construct was then introduced into MG1655Z1 (9Bohn C. Collier J. Bouloc P. Mol. Microbiol. 2004; 52: 427-435Crossref PubMed Scopus (34) Google Scholar) by P1 vir-mediated transduction, resulting in strain PhB3089. To overexpress RydC, the rydC gene was PCR-amplified using P5 (5′-CGGAATTCCGGCATCGGCATAAAAGG-3′) and P6 (5′-CGGGATCCTCTGGAAGGACAACACAC-3′). The resulting fragment was digested by BamHI and EcoRI and cloned into BamHI-EcoRI restricted pUC18, resulting in strain PhB3203. Constructions were verified on an ABI310 automatic DNA sequencer (Applied Biosystems).RNA Isolation and Northern Blots—E. coli strains were grown in either Luria-Bertani (LB) or minimal media (M9) and harvested at the indicated A600. The cell pellets were resuspended in Trizol (Invitrogen). Total RNA extraction was performed as suggested by the manufacturer. Total RNAs were isolated either by Trizol reagent (Invitrogen) or by acid-phenol extraction, in which the cell pellet was dissolved in 0.2 m sodium acetate, 10 mm EDTA, 1% SDS (pH 5.0), with a volume of water-saturated phenol pre-heated at 65 °C. Incubation was performed for 10 min at 65 °C and 0.5 volume of CHCl3:isoamylic alcohol (24:1). The sample was incubated for 10 additional minutes at 65 °C. Total RNAs were precipitated overnight at 4 °C with 2 m LiCl (final concentration), washed with 80% ethanol, and incubated with an RNase-free DNase followed by phenol extraction. For RydC and transfer-messenger RNA, Northern blots were carried out by loading 30 μg of freshly extracted RNAs per lane onto an 8% PAGE in 1× Tris-borate EDTA for 3 h at 350 V. For yejABEF mRNA, we used a 1% agarose gel in 2.2 m formaldehyde, in 20 mm MOPS (pH 7.0), 8 mm sodium acetate, and 1 mm EDTA (pH 8.0). For the blots performed on polyacrylamide gels, the transfer of the RNAs was achieved in 0.5× Tris-borate EDTA onto charged nylon membranes (Zeta probe GT; Bio-Rad) for 3 h at 20 V at 4 °C. For the blots performed on agarose gels, the transfer of the RNAs was achieved in 20× SSC onto neutral nylon membranes overnight at room temperature (passive transfer). Either 100–1000- or 200–10000-nt-long RNA markers (Novagen) were used. To monitor yejABEF expression in vivo in a wild-type strain, ΔRydC, and pUC-RydC strains, primer P7 (5′-GTATCGATACCGAGGCTATAGATGGGGC-3′) was used for hybridization. To monitor RydC expression in vivo in a wild-type strain, ΔRydC, and pUC-RydC strains, primer P8 (5′-ACCGACCCGTGGTACAGGCG-3′) was used for hybridization.Primer Extension and RACE—Total RNAs (15 μg) were annealed to 5′-labeled primer P8 at 90 °C for 30 s and 65 °C for 5 min, cooled down on ice, and subjected to primer extension at 42 °C for 30 min with 5 units of avian myeloblastosis virus reverse transcriptase (Qbiogene) and 0.5 mm deoxynucleotide triphosphates in a volume of 5 μl. The cDNA products were separated on a 6% PAGE. RACE assays were carried out according to Ref. 10Argaman L. Hershberg R. Vogel J. Bejerano G. Wagner E.G. Margalit H. Altuvia S. Curr. Biol. 2001; 11: 941-950Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, with modifications: for the 5′-RACE, 10 μg of total RNAs, with and without a treatment with tobacco acid pyrophosphatase, was denatured in presence of 300 pmol of RNA adapter R1 (5′-UGGCGGACGCGGGUUCAACUCCCGCCAGCUCCACCA-3′) (Dharmacon) at 95 °C for 3 min and cooled down on ice. Ligation was performed at 16 °C overnight with 10 units of T4 RNA ligase (New England Biolabs) in 50 mm Tris-HCl (pH 7.9), 10 mm MgCl2, 4 mm dithiothreitol, 150 μm ATP, and 10% Me2SO. After phenol chloroform extraction and ethanol precipitation, 2.5 μg of RNA was reverse-transcribed with 2 pmol of gene-specific complementary DNAs P8 or P9 (5′-GTACAGGCGAAGAATACGGG-3′) with 20 units of avian myeloblastosis virus reverse transcriptase (Qbiogene) at 42 °C for 30 min in a final volume of 20 μl. 1 μl of the reaction was used for PCR amplification by 15 pmol of primer P8 or P9 and adapter-specific homologous primer P10 (5′-AACTCCCGCCAGCTCCACCA-3′), 1 unit of Taq DNA polymerase (New England Biolabs) in 2.5 mm MgCl2, and 0.2 m of each deoxynucleotide triphosphate. Products were separated on a 10% PAGE, eluted, and cloned into PCR2.1 TOPO vector (Invitrogen). Recombinant bacteria were selected by PCR, and the positive ones were sequenced by M13 reverse and universal DNA primers. For the 3′-RACE, calf intestinal phosphatase-treated total RNA was ligated to 5′-phosphorylated R1. Reverse transcription was performed with 200 pmol of primer P11 (5′-AGTTGAACCCGCGTCCGCCA-3′) complementary to R1. PCR amplification was performed with gene-specific DNA P12 (5′-GATGTAGACCCGTATTCTTCG-3′), as described for the 5′-RACE.In Vitro Transcription and RNA Labeling—RydC and yejAB were PCR-amplified from genomic DNA. Forward primers contain a T7 promoter sequence and a BamHI site. For RydC, the forward primer was P13 (5′-CGGGATCCTAATACGACTCACTATAGGGCTTCCGATGTAGACCCGTAT-3′) and the reverse primer was P14 (5′-AAGAAAACGCCTGTACTAAAAC-3′). For yejAB, the forward primer was P15 (5′-TAATACGACTCACTATAGGGCCCCATCTATAGCCTCGGTATCG-3′) and the reverse primer was P16 (5′-ATCACCAGCAACAGACGGCG-3′). Transcription mixtures contained PCR-generated DNA templates (0.02 μm), 0.5 mm of the four ribodeoxynucleotides (ATP, GTP, UTP, and CTP), 2.5 units/μl T7 RNA polymerase (Invitrogen) in 80 mm Tris-HCl (pH 7.9), 20 mm dithiothreitol, 20 mm NaCl, 2 mm spermidine, and 12 mm MgCl2. The incubation was for 1 h at 37 °C, and then 5 units of DNase I was added for 15 min, followed by phenol extraction and ethanol precipitation. Labeled transcriptions of yejAB were performed the same way, except that the final concentration of cold UTP was reduced to 0.01 mm and [α-32P]UTP was added (800 Ci/mmol; Amersham Biosciences). Labeled and unlabeled RNAs were purified on a 10% PAGE. RydC was radiolabeled at either its 5′-end using [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (Invitrogen) or its 3′-end using [α-32P]pCp and T4 RNA ligase (New England Biolabs) and purified on a 10% PAGE.Gel-shift Assays—Hfq was overproduced from strain BL21(DE3) transformed with pTE607 plasmid (kindly provided by Dr. Hajnsdorf, Institut de Biologie Physico-Chimique, Paris, France). E. coli BL21(DE3) cells were grown in 3 liters of LB medium at 37 °C to an A600 of 0.4, induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h, and centrifuged for 20 min at 5000 × g (4 °C). The bacterial pellets were dissolved in 20 mm Tris-HCl (pH 7.8), 500 mm NaCl, 10% glycerol, and 0.1% Triton X-100 in the presence of protease inhibitor mixture (Roche Applied Science) and sonicated on ice (6 rounds of 10 s each with 10-s pauses), followed by treatment with DNase I (100 units/ml, 15 min, 37 °C) and a 20-min centrifugation (10,000 × g at 4 °C). The supernatant was filtered (0.45 μ), loaded onto a FPLC (Amersham Biosciences) equipped with a Ni2+ column washed with 50 mm NaH2PO4, 300 mm NaCl, and 10 mm imidazole; equilibrated at pH 6.0; and eluted in a similar buffer, except for the concentration of imidazole (300 mm). The protein was pure, as shown on a 12% SDS-PAGE (the monomeric and multimeric forms of Hfq were visible, even after boiling the sample in SDS buffer), heated for 15 min at 80 °C, and centrifuged for 10 min at 13,000 × g at room temperature, and the supernatant was concentrated on 5kD Amicon in 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 50 mm NH4Cl, 5% glycerol, and 0.1% Triton X-100. Protein concentration was determined using the Bradford assay, and the protein was stored at 4 °C.The transcripts were denatured in 50 mm Hepes (pH 6.9), 50 mm NaCl, 5 mm KCl, and 1 mm MgCl2 for 3 min at 85 °C, followed by refolding for 10 min at 30 °C and chilled on ice. 0.02 to 1 pmol of labeled RNA was incubated with a 1000–1500 molar excess of carrier yeast tRNAs, 0.2 μg/μl bovin serum albumin, and the indicated amounts of purified Hfq. The binding reactions were in 10 mm Tris-HCl (pH 7.5), 6 mm NaCl, 10 mm EDTA, and 5 mm dithiothreitol for 10 min at 30 °C. The samples were supplemented with 10% glycerol (final concentration) and loaded on a native 4% polyacrylamide gel containing 5% glycerol. The electrophoresis was performed in 0.5× Tris-borate EDTA supplemented with 0.5% glycerol at 4 °C for 4 h (100 V). The results were analyzed and quantified either on a PhosphorImager (Amersham Biosciences) or directly by autoradiography.Isolation of RNAs That Bind to a RydC-Hfq Complex—Total RNAs were extracted at A600 = 0.4. Both the 16S and the 23S rRNAs were removed (Single Place Magnetic Stand; Ambion). RNAs (10 μg) depleted in both 16S and 23S rRNAs, corresponding to roughly 30 μg of total RNAs, were 3′-end-labeled (as described for RydC, see above) and purified from the unincorporated [α-32P]pCp using MicroSpin™ G-25 columns. A complex between 1.5 pmol of refolded cold RydC and 37.5 pmol (a 25-fold molar excess) of His-tagged Hfq was formed in 10 mm Tris-HCl (pH 8.0), 50 mm NaCl, 50 mm KCl, and 10 mm MgCl2 at 37 °C for 15 min (at that ratio, all RydC was bound to Hfq). 3′-labeled bacterial RNAs were added to the RydC-Hfq complex, incubated for an additional 15 min at 37 °C, and loaded on a 0.5 ml TALON® metal affinity column (Clontech) in 20 mm Tris-HCl (pH 7.8), 500 mm NaCl, 10% glycerol, and 0.1% Triton X-100. The elution was performed in three steps. In each, 400 μl of 50 mm NaH2PO4 (pH 6.0), 300 mm NaCl, and 300 mm imidazole was added to the column (first at 37 °C for 30 min, and then at 37 °C for 60 min) and, finally, 400 μl of 50 mm NaH2PO4 (pH 6.0), 300 mm NaCl, and 300 mm imidazole was added to the column, and the resin was treated with an equal volume of phenol (pH 4.5). The three fractions were collected, treated with phenol/chloroform, and ethanol-precipitated. The eluted RNAs were cloned as described for the 3′-RACE. The terminal 5′-P of the eluted RNAs was removed by the calf intestinal phosphatase. The eluted RNAs are 3′-end-labeled with [α-32P]pCp, thus a 5′-OH R1 can be ligated at their 3′-ends, and cDNAs were generated by primer extension with P11. R1 phosphorylated at its 5′-end was then ligated at the 3′-ends of the cDNAs, PCR-amplified, and loaded onto a 10% PAGE. The PCR fragments were eluted individually, re-amplified by PCR, cloned, and sequenced.Structural Analyses—End-labeled and gel-purified RydC were folded in 50 mm Hepes (pH 6.9), 50 mm NaCl, 50 mm KCl, and 1 mm MgCl2 for 3 min at 85 °C and then folded for 10 min at 30 °C and chilled on ice. For secondary structure analysis, the RNAs were partially digested at 30 °C for 10 min with 10–4 or 5.10–5 unit of RNase V1 in 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, and 50 mm KCl with 2.5 μg of yeast tRNAs. Limited digestions with 0.2 and 1 unit of nuclease S1 were performed at 30 °C for 10 min in 25 mm sodium acetate (pH 4.5), 10 mm MgCl2, 50 mm KCl, and 1 mm ZnCl2 with 2.5 μg of yeast tRNAs. Partial alkaline hydrolysis was performed in 0.1 m NaOH and 2 mm EDTA at 90 °C for 50 s. RNase T1 sequencing was performed in 20 mm sodium acetate (pH 5.0), 1 mm EDTA, and 7 m urea at 55 °C for 6 min in the presence of 10 μg of yeast tRNAs with 2 units of RNase T1 (final volume, 5 μl). The samples were directly loaded on a 12% PAGE. Probing the structure of either 5′- or 3′-end-labeled RydC in complex with Hfq was performed after refolding the RNA as described above. Then, RydC was bound to a 50-fold molar excess of Hfq. The complex was partially digested at 30 °C for 7 min with either 10–4 or 5.10–5 unit of RNase V1 or 0.5 unit to 5 units of nuclease S1 supplemented with 1 mm ZnCl2. There was either a 400- or a 1200-fold molar excess of yeast tRNA when probing the conformation of RydC in complex with Hfq.RESULTSIdentification, Expression, and End Mapping of a Novel sRNA—Based on sequence conservations between phylogenetically related species, a computer approach was developed (11Pichon C. Felden B. Bioinformatics. 2003; 19: 1707-1709Crossref PubMed Scopus (40) Google Scholar) to identify novel sRNAs expressed from the intergenic regions of the E. coli genome. All those previously characterized (1Masse E. Majdalani N. Gottesman S. Curr. Opin. Microbiol. 2003; 6: 120-124Crossref PubMed Scopus (121) Google Scholar) were identified, plus new ones. Among them, –10 and –35 promoter sequence signals flanked at their 3′-sides by a 50–60-nt-long “GC-rich” region ending by a putative “Rho-independent” terminator could be predicted (Fig. 1A). We selected one of these putative sRNA-encoding genes that was identified in 21 sequenced bacterial genomes, all from the family of the enterobacteriaceae (6 E. coli, 11 Salmonella, and 4 Shigella sequences), for additional studies. A sequence alignment of those that possess sufficient sequence variations is presented in Fig. 1A. While our work was in progress, the presented putative E. coli sRNA was isolated by co-immunoprecipitation with Hfq, identified using microarray and Northern blot analysis, and named RydC (6Zhang A. Wassarman K.M. Rosenow C. Tjaden B.C. Storz G. Gottesman S. Mol. Microbiol. 2003; 50: 1111-1124Crossref PubMed Scopus (429) Google Scholar), the name by which we will refer to it. The RydC-encoding gene, rydC, is located at 32 min on the E. coli genetic map between cybB and ydcA encoding cytochrome b561 and a hypothetical protein of 5.9 kDa, respectively, both located on the complementary DNA strand (Fig. 1B).Based on the alignment of seven sequences with high nucleotide identity, RydC is proposed to fold as a pseudoknot, as confirmed experimentally using structural probes (see below). Two RNA helices H1 (7 bp) and H2 (9 bp) are predicted. H2 is part of a Rho-independent terminator. Divergent rydC sequences possess three compensatory mutations to maintain the pairings within RNA helix H2. Predicted helices H1 and H2 are entangled and connected by three nt stretches L1, L2, and L3. Based on the known genes, L1 has 6–7 “pyrimidine-rich” nt, L2 has a single conserved Cys residue, and L3 has 7–8 nt.Primer extension analysis on total cellular RNA demonstrates that the predicted promoter is functional in vivo (Fig. 1C). Determination of the 5′ and 3′ boundaries of RydC was required for subsequent functional and structural analyses. They were determined using 5′- and 3′-RACE (Fig. 1D). From several DNA clones, RydC 5′-end mapped at two adjacent positions (Fig. 1A, boxed 5′-CT) 5 or 6 nucleotides downstream from the “–10 box” of the predicted promoter. RydC 3′-end also mapped at two adjacent positions (Fig. 1A, boxed 3′-TT or TG), downstream from the 3′-strand of H2 ending by a T4C stretch that forms a Rho-independent terminator. As determined from the major 5′- and 3′-end points, RydC has 62–64 nucleotides in vivo, consistent with the size estimated from our computer prediction.RydC Forms a Complex with Hfq—Because RydC interacts with Hfq in vivo (6Zhang A. Wassarman K.M. Rosenow C. Tjaden B.C. Storz G. Gottesman S. Mol. Microbiol. 2003; 50: 1111-1124Crossref PubMed Scopus (429) Google Scholar), we hypothesized that its quantity could be Hfq-dependent. RydC expression was compared by Northern blots between a strain deficient for Hfq (Δhfq) and its parental strain. In contrast to the parental strain, RydC could not be detected in the derivative that does not express Hfq (Fig. 2A). It suggests that in the absence of Hfq, RydC is unstable and rapidly degraded by RNases. Stability of sRNAs can be reduced in the absence of Hfq that protects them against RNase activity, including RNase E (1Masse E. Majdalani N. Gottesman S. Curr. Opin. Microbiol. 2003; 6: 120-124Crossref PubMed Scopus (121) Google Scholar, 5Moll I. Afonyushkin T. Vytvytska O. Kaberdin V.R. Blasi U. RNA (N. Y.). 2003; 9: 1308-1314Crossref PubMed Scopus (208) Google Scholar). Alternatively, Hfq might be required for the transcription of RydC. The binding of Hfq to RydC was tested in vitro by gel-shift assays using increasing amounts of purified Hfq and constant amounts of labeled synthetic RydC. Binding assays were performed at various salt conditions, constant pH (pH 7.5–8.0), and a large excess of bulk tRNA from yeast to reduce aspecific binding (from a 30 to a 1500 molar excess relative to RydC). The apparent binding constant between RydC and Hfq varies from 120 to 500 nm, depending on the amount of carrier tRNA (the lowest association constant corresponds to the lowest molar excess of competitor RNA). Retardation assays with increasing amounts of purified Hfq indicate that the RNA-protein complex is detected in vitro at physiological pH and salt concentrations (Fig. 2B shows a typical assay). The binding between RydC and Hfq is specific because a 4000-fold molar excess of total tRNA displaces only a minor fraction of RydC from a preformed RydC-Hfq complex, whereas a 50-fold excess of cold RydC competed labeled RydC out of the complex (Fig. 2C).Fig. 2Stabilization of RydC by Hfq in vivo and assessment of the affinity and specificity of the interaction. A, detection of RydC expression by Northern hybridization using a labeled strand-specific probe during bacterial growth in LB medium in cells that do not express Hfq because of chromosomal gene disruption (Hfq Δ) compared with a control strain (Hfq wt). B, native gel retardation assay of purified labeled RydC with increasing amounts of purified His-tagged Hfq. C, the interaction between Hfq and RydC is specific. Native gel retardation assay of labeled RydC and Hfq in the presence of increasing amounts of either unlabeled total E. coli tRNA or unlabeled RydC.View Large Image Figure ViewerDownload (PPT)Structural Analysis of RydC and Its Interaction with Hfq— Sequence variation is too low (the sequences aligned have a high sequence identity) to fully establish the secondary structure of RydC by a phylogenetic analysis. Therefore, its conformation was analyzed further by structural probes in solution, an approach that was instrumental in establishing the secondary structures for many RNAs (12Brunel C. Romby P. Methods Enzymol. 2000; 318: 3-21Crossref PubMed Google Scholar). A RydC transcript was end-labeled, and its solution conformation was probed by enzymes. RNase V1 cleaves double-stranded RNA or stacked nucleotides, whereas nuclease S1 cleaves single-stranded RNA. The reactivity toward these probes was monitored for each nucleotide of a 64-nt-long synthetic RNA, in the absence and presence of the protein Hfq. Four independent experiments were performed on RydC alone, and four additional ones were performed on the Hfq-RydC complex (Fig. 3, A–C, is representative). These data are summarized on secondary structure models that they, together with the phylogenetic analysis, support (Fig. 3, D and E).Fig. 3Monitoring the solution conformation of RydC (A and D) and the RydC-Hfq complex (B, C, and E) by structural probes. A–C, autoradiograms of 12% PAGE separation of 5′ (A and C)- or 3′ (B)-labeled RydC fragments generated by single (nuclease S1)- or double (RNase V1)-strand-specific digestions in the absence (A) or presence (B and C, +) of Hfq. The secondary structural elements of the RNA are indicated. RNase T1 hydrolysis ladders (lanes GL) indicate the position of each guanosine residue within the RNA sequence (left). Hydrolysis ladders (lanes L) indicate the position of each residue along the RNA sequence. D, experimentally supported secondary structure of RydC. H1 and H2 are the helices; L1 and L3 are the loops crossing the major and minor grooves of the RNA structure, respectively; and L2 connects H2 to H1. Arrowheads show the V1 cleavages, stars show the S1 cuts. Black symbols represent strong cleavage, gray symbols represent moderate cleavage, and white symbols represent weak cleavage. E, enzymatic footprints of RydC in the presence of Hfq. There is a 50:1 molar ratio of the protein to the RNA, with a 1200 molar excess of carrier tRNA. Nucleotides whose reactivity is unaffected upon protein binding are represented by gray circles. Nucleotides with modified reactivity in the presence of Hfq are in capital letters. Symbols are as defined in D. Enzymatic cleavages that are reduced (–) or enhanced (+) upon protein binding are indicated. Upon Hfq binding, five alternate pairings between nt located within the two black boxes are proposed.View Large Image Figure ViewerDownload (PPT)Double-stranded-specific cuts from C15 to U18 and at the predicted G12-C41 pair and the absence of nuclease S1 cleavages at G12-U18 and A35-C41 suggest that helix H1 forms in solution (Figs. 1A and 3D). RNase V1 cuts at U29-U31, G54, and G57 and the absence of S1 cleavages between C25-C33 and G49-G57 support the existence of a 9-bp helix H2 (Figs. 1A and 3D). S1 cleavages at A19-U23 and U44-U47 are consistent with loops L1 and L3 being mostly single-stranded in solution. According to the sequences, the nt content of loop L1 varies from six to seven, and that of L3 varies from seven to eight. L1 and L3 cross the deep and shallow grooves of the RNA structure that folds as a pseudoknot (13Draper D.E. Trends Biochem. Sci. 1996; 21: 145-149Abstract Full Text PDF PubMed Scopus (107) Google Scholar). A conserved unpaired Cys residue is in between helices H1 and H2 that forms L2. L2 is not cut by S1, probably because it is not accessible for cleavage. The strong S1 cleavages between U58 and U63 suggest that the “uridine-rich” 3′-end of RydC is unpaired. Nucleotides C5 and G6 are cleaved by both single-stranded- and double-stranded-specific probes, suggesting that the 5′-end of RydC breathes in solution, as for other bacterial RNAs (14Felden B. Himeno H. Muto A. McCutcheon J.P. Atkins J.F. Gesteland R.F. RNA (N. Y.). 1997; 3: 89-104PubMed Google Scholar). Alternatively, the nt segment 4CCGAU8 can pair transiently with 39GUCGG43 (Fig. 3D, boxed nt), accounting for the V1 cuts at nt C5 and G6, forming an extended helix interrupted by an internal bulge at nt G9-C14. There are no detectable degradation sites within the sequence of RydC.The interaction between RydC and Hfq was also monitored by structural probes (Fig. 3, B, C, and E). In the presence of Hfq, nuclease S1 cleavages disappear at U21-U24 in loop L1 and at U44-U47 in loop L3. These regions are accessible “uridine-rich”“sequences. Because Hfq binds “uridine-rich” sequences close to structured domains in target RNAs (15Zhang A. Wassarman K.M. Ortega J. Steven" @default.
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