Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator

Abstract

The Sm-like protein Hfq is involved in post-transcriptional regulation by small, noncoding RNAs in Escherichia coli that act by base pairing. Hfq stabilises the small RNAs and mediates their interaction with the target mRNA by an as yet unknown mechanism. We show here a novel chaperoning use of Hfq in the regulation by small RNAs. We analysed in vitro and in vivo the role of Hfq in the interaction between the small RNA RyhB and its sodB (iron superoxide dismutase) mRNA target. Hfq bound strongly to sodB mRNA and altered the structure of the mRNA, partially opening a loop. This gives access to a sequence complementary to RyhB and encompassing the translation initiation codon. RyhB binding blocked the translation initiation codon of sodB and triggered the degradation of both RyhB and sodB mRNA. Thus, Hfq is a critical chaperone in vivo and in vitro, changing the folding of the target mRNA to make it subject to the small RNA regulator.

Figure 1

Hfq binding to sodB and RyhB RNA. (A, B) In all, 0.1 nM of [α-³²P]UTP-labelled sodB_1–148 (A) or RyhB (B) transcript was incubated without or with various concentrations of purified Hfq (indicated above the gel) in the presence of 100 ng/μl tRNA. After incubation for 5 min at 37°C, the mixture was analysed by electrophoresis in a native polyacrylamide gel.

Figure 2

Binding of sodB mRNA and competitors to Hfq. (A) Secondary structure of the 5′ end of sodB mRNA, as predicted by the mfold program. Transcription begins at nucleotide 1 and translation is initiated at nucleotide 56. The AUG start codon is shown in bold. The numbering indicates the positions of the truncated sodB RNA fragments used as competitors in gel mobility shift assays. The nucleotides exchanged during construction of the mutations in the A/T-rich region (AT) or of the Shine–Dalgarno sequence (s.d.) are shown in bold. The stem-loops of sodB are indicated by lowercase letters in bold typeface. (B) Gel mobility shift assay with truncated sodB mRNA fragments competing against sodB_1–148 RNA for Hfq binding. Labelled sodB_1–148 transcript was incubated with (+) or without (−) Hfq protein. Unlabelled competitor RNA was added at 5000-fold molar excess. The competitor added in each case is indicated at the top of the lane.

Figure 3

Minimal requirements for sodB and RyhB RNA binding to Hfq. (A, B) Minimal binding assay. 5′ or 3′ end-labelled sodB_1–148 RNA (A) and RyhB RNA (B) was subjected to partial alkaline hydrolysis. The resulting fragments were then incubated with Hfq, and bound and unbound fragments were separated by native polyacrylamide electrophoresis and fractionated on a denaturing polyacrylamide gel. In the experiments with 3′ end-labelled RNAs, two different complexes of bound RNA were detected and fractionated separately. The numbers to the left indicate sequence positions with respect to the transcription start site. (C, D) Summary of the minimal binding assay. The minimal 5′ and 3′ end-labelled sodB_1–148 (C) and RyhB (D) RNA sequences that bound to Hfq are shown. The stem-loops of sodB are indicated by lowercase letters in bold typeface, and the stem-loops of RyhB are indicated by numbers in bold typeface.

Figure 4

Changes in RNA structure upon Hfq binding. (A) RNase footprinting. 5′ end-labelled sodB_1–148 (A) or RyhB (B) transcript was subjected to partial digestion with RNase A, RNase T₁ or RNase V₁, with (+) or without (−) purified Hfq protein. In (A), 3′ end-labelled sodB_1–148 was digested with RNase I. The resulting fragments were then analysed on a denaturing sequencing gel. The numbers to the left indicate sequence positions with respect to the transcription start site. (C, D) Summary of the RNase footprints of sodB_1–148 (C) and RyhB (D) RNA. Elongated triangles indicate RNase A cleavage sites. Arrowheads indicate RNase T₁ cleavage sites, and asterisks indicate RNase V₁ cleavage sites. Nucleotides for which cleavage was more or less likely to occur in the presence of Hfq are shown in green and red, respectively. Circled residues indicate unusual cleavage sites for RNase T₁ upon Hfq binding. Secondary structures were predicted with the mfold program, based on the results of footprinting experiments.

Figure 5

RyhB–sodB RNA interaction in the presence of Hfq. (A) RNase footprinting. In all, 0.2 pmol of 5′ end-labeled sodB_1–148 transcript was incubated with Hfq (1 pmol) and then subjected to partial digestion with RNase A, RNase T₁ or RNase V₁, without or with unlabelled RyhB RNA (0, 1, 3 or 5 pmol). The resulting fragments were then analysed on a denaturing sequencing gel. The numbers to the left indicate sequence positions with respect to the transcription start site, and the black vertical line indicates the region protected upon base pairing. (B) RNase footprinting. In all, 0.2 pmol of 5′ end-labelled RyhB transcript was incubated with Hfq (1 pmol) and subjected to partial digestion with RNase A, RNase T₁, RNase V₁ or RNase I, with or without unlabelled sodB_1–148 or full-length sodB RNA (5 pmol). The resulting fragments were then analysed on a denaturing sequencing gel. The numbers to the left indicate sequence positions with respect to the transcription start site and the black vertical line indicates the region protected upon base pairing.

Figure 6

Role of Hfq in sodB expression and on sodB mRNA stability. (A) Strains fur⁺ and fur⁻, carrying the fusion (sodB-lacZ)₁₉ (black) and the fusion (sodB-lacZ)₁₈ (red), were grown in LB medium and assayed for β-galactosidase activity. β-Galactosidase activity, expressed in Miller units per millilitre, is plotted against OD₆₀₀ units. The values shown are representative of three experiments for which individual values did not differ by more than 15%. (sodB-lacZ)₁₉: □, wild type; ▪, fur; ○, hfq; •, fur hfq; (sodB-lacZ)₁₈: ▵, wild type; ▴, fur. (B) Strains containing plasmid pHS1–8 were grown at 37°C to an OD of about 1. Rifampin was added to a final concentration of 150 μg/ml, and samples were taken following incubation at 37°C for Northern analysis. Half-lives estimated from quantitative analysis were: 17.5 min in the wild type and hfq strains; 15.5 min in the fur hfq strain; and 4.75 min in the fur strain.

Figure 7

Model for sodB mRNA–Hfq–RyhB interaction. Hfq binds with high affinity to sodB mRNA, via an A/U-rich sequence preceding stem-loop b. This binding causes the mRNA to adopt a structure in which stem-loop b, which follows the Hfq-binding site, is opened out to give a large loop containing the translation start codon, which lies within the sequence complementary to RyhB. The stem of stem-loop b starts with the ribosome-binding site. In conditions of iron deficiency (Fur inactivated), RyhB is produced and is stabilised by binding to Hfq. RyhB interacts with sodB mRNA by base pairing in the region containing the complementary sequence. This base pairing both modifies the structure of the RNA molecule and blocks translation. Changes in the structure of stem-loop b may lead to the release of Hfq. The block of translation and the structural change render the RNA molecule susceptible to RNase cleavages. Numbering starts at the transcription start site. The translation start site of sodB is indicated by an arrow. Hfq-binding sites are shown in red, and sequences complementary between sodB and RyhB are shown in green. Regions affected by Hfq binding are shown in bold.