Translational autocontrol of the Escherichia coli hfq RNA chaperone gene

Abstract

The conserved bacterial RNA chaperone Hfq has been shown to play an important role in post-transcriptional regulation. Here, we demonstrate that Hfq synthesis is autoregulated at the translational level. We have mapped two Hfq binding sites in the 5'-untranslated region of hfq mRNA and show that Hfq binding inhibits formation of the translation initiation complex. In vitro translation and in vivo studies further revealed that Hfq binding to both sites is required for efficient translational repression of hfq mRNA.

FIGURE 1.

Hfq has two binding sites in the 5′-UTR of hfq mRNA. (A) Primary structure of the hfq 5′-UTR and the 5′-initial coding region containing two stem–loop structures h1 and h2 (see D). The SD sequence and the start codon are underlined. Regions protected by Hfq from hydroxyl radical cleavage (see C) are shown by dotted lines below the sequence. The binding site of the F22 oligonucleotide used in the toeprinting assay shown in Figure 2C is indicated. The hfq126, hfq96, hfq76, and hfq46 mRNA fragments used in the gel-mobility shift assays shown in B are depicted by black lines. The 5′-terminal portions of the hfqΔ30 mRNA used for in vitro translation and that of the hfq131-lacZ and hfq101-lacZ fusion genes are also shown. (B) Hfq binding to hfq126, hfq96, hfq76, and hfq46 mRNA fragments. 5′-end labeled transcripts (5 nM) were incubated in the absence (lane 1) or in the presence of increasing molar ratios of Hfq. The Hfq-hexamer (Hfq₆) concentrations in panel I were 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM (lanes 2–6) and in panels II–IV were 5 nM, 10 nM, 20 nM, 30 nM, and 50 nM (lanes 2–6). (C) Hydroxyl radical footprints of hfq126 mRNA in the presence of Hfq. Samples containing 5′-end-labeled hfq126 mRNA were incubated in the absence (lane 3) or in the presence of Hfq (lanes 4,5) and subjected to hydroxyl radical cleavage. Hfq was added in eightfold (lane 4) and 16-fold (lane 5) molar excess (Hfq₆ excess) over hfq126 mRNA. Lane 1, untreated hfq126 mRNA (5 nM) was incubated with Hfq (80 nM Hfq₆). Lane 2, RNase T1 cleavage (G-specific cleavage) of hfq126 mRNA. The regions protected from hydroxyl-radical cleavage by Hfq (sites A and B) are marked by bars (see also A). Nucleotide positions are given at the left. (D) RNase T1 probing of secondary structures within the 5′-UTR and 5′-initial coding region of hfq mRNA. The hfq mRNA was hybridized to 5′-end labeled primer Y19 and then subjected to RNase T1 cleavage. The cleavage sites were mapped by primer extension. The reactions were incubated in the absence (lane 7) or in the presence of 0.1 U (lane 5) and 0.5 U (lane 6) of RNase T1, respectively. The G residues protected from RNase T1 cleavage are numbered relative to the A (+1) of the start codon of hfq mRNA and are marked by open arrowheads. Regions corresponding to stem–loop structures h1 and h2 are depicted by bars at the left. Lanes 1–4, sequencing reactions.

FIGURE 1.

Hfq has two binding sites in the 5′-UTR of hfq mRNA. (A) Primary structure of the hfq 5′-UTR and the 5′-initial coding region containing two stem–loop structures h1 and h2 (see D). The SD sequence and the start codon are underlined. Regions protected by Hfq from hydroxyl radical cleavage (see C) are shown by dotted lines below the sequence. The binding site of the F22 oligonucleotide used in the toeprinting assay shown in Figure 2C is indicated. The hfq126, hfq96, hfq76, and hfq46 mRNA fragments used in the gel-mobility shift assays shown in B are depicted by black lines. The 5′-terminal portions of the hfqΔ30 mRNA used for in vitro translation and that of the hfq131-lacZ and hfq101-lacZ fusion genes are also shown. (B) Hfq binding to hfq126, hfq96, hfq76, and hfq46 mRNA fragments. 5′-end labeled transcripts (5 nM) were incubated in the absence (lane 1) or in the presence of increasing molar ratios of Hfq. The Hfq-hexamer (Hfq₆) concentrations in panel I were 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM (lanes 2–6) and in panels II–IV were 5 nM, 10 nM, 20 nM, 30 nM, and 50 nM (lanes 2–6). (C) Hydroxyl radical footprints of hfq126 mRNA in the presence of Hfq. Samples containing 5′-end-labeled hfq126 mRNA were incubated in the absence (lane 3) or in the presence of Hfq (lanes 4,5) and subjected to hydroxyl radical cleavage. Hfq was added in eightfold (lane 4) and 16-fold (lane 5) molar excess (Hfq₆ excess) over hfq126 mRNA. Lane 1, untreated hfq126 mRNA (5 nM) was incubated with Hfq (80 nM Hfq₆). Lane 2, RNase T1 cleavage (G-specific cleavage) of hfq126 mRNA. The regions protected from hydroxyl-radical cleavage by Hfq (sites A and B) are marked by bars (see also A). Nucleotide positions are given at the left. (D) RNase T1 probing of secondary structures within the 5′-UTR and 5′-initial coding region of hfq mRNA. The hfq mRNA was hybridized to 5′-end labeled primer Y19 and then subjected to RNase T1 cleavage. The cleavage sites were mapped by primer extension. The reactions were incubated in the absence (lane 7) or in the presence of 0.1 U (lane 5) and 0.5 U (lane 6) of RNase T1, respectively. The G residues protected from RNase T1 cleavage are numbered relative to the A (+1) of the start codon of hfq mRNA and are marked by open arrowheads. Regions corresponding to stem–loop structures h1 and h2 are depicted by bars at the left. Lanes 1–4, sequencing reactions.

FIGURE 2.

Hfq represses translation of its own mRNA. (A) Hfq inhibits in vitro translation of hfq mRNA. Equimolar concentrations (100 nM) of full length hfq and ppiB mRNAs were used for in vitro protein synthesis as described in Materials and Methods. The translation reactions were carried out in the absence (lane 3) and in the presence (lanes 4–6) of increasing molar amounts of Hfq to both mRNAs as indicated on top of the autoradiograph (ratios correspond to molar excess of Hfq₆ over totally added mRNA). Lanes 1 and 2, in vitro translation of hfq and ppiB mRNA in the absence of Hfq, respectively. The positions of the [¹⁴C]-labeled Hfq and PpiB proteins are indicated by arrowheads. (B) Autogenous inhibition of translation initiation by Hfq. The toeprinting reactions were performed as described in Materials and Methods. Lane 1, primer extension in the absence of 30S subunits, tRNA_f^Met, and Hfq. Lane 2, toeprinting in the presence of 30S subunits, tRNA_f^Met, and absence of Hfq. Lanes 3–5, toeprinting with 30S subunits, tRNA_f^Met, and in the presence of increasing molar Hfq-hexamer ratios to hfq mRNA as indicated on top of the autoradiograph. Hfq was added prior to the addition of ribosomes. The arrowhead depicts the toeprint signal at position +15 relative to the A of the ATG start codon. The relevant part of the DNA sequence of the hfq 5′-UTR is shown at the right. The position of the Shine and Dalgarno (SD) sequence and of the ATG start codon is indicated. (C) Lack of ribosome binding to hfq mRNA annealed to F22 oligonucleotide. In vitro toeprinting assay was performed on hfq mRNA after annealing of oligonucleotide F22 as described in Materials and Methods. Lane 1, primer extension (Ext) in the absence of 30S subunits, tRNA_f^Met, and Hfq. Lane 2, toeprinting in the presence of 30S subunits, tRNA_f^Met, and in the absence of Hfq. Lane 3, toeprinting with 30S subunits, tRNA_f^Metin the presence of a 20-fold molar excess of Hfq (Hfq_f^Metratio). The arrow indicates the position of the toeprint signal(s). (D) The relative translational efficiency of the hfq131-lacZ gene depends on Hfq. The expression of hfq131-lacZ fusion was induced as described in Materials and Methods. The averaged β-galactosidase values normalized to mRNA levels obtained in the hfq⁻ strain (control) was set to 1 (white bar). The value obtained with the hfq⁺ strain (gray bar) was normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations.

FIGURE 3.

Both Hfq binding sites are necessary for efficient translational repression of hfq mRNA. (A) In vitro translation of hfq wild-type and hfqΔ30 mRNAs. Equimolar concentrations (100 nM) of hfq and hfqΔ30 mRNAs were used. The translation reactions with the individual mRNAs were carried out in the absence (lane 1) or in the presence of increasing molar amounts of Hfq. The protein was added to the reactions at molar Hfq₆ ratios of 0.5:1 (lane 2), 1:1 (lane 3), 2:1 (lane 4), 3:1 (lane 5), 4:1 (lane 6), 5:1 (lane 7), and 6:1 (lane 8) to hfq mRNA(s). The [¹⁴C]-labeled translation products were resolved by SDS-PAGE, and the gels were subjected to autoradiography. A graphical representation of three independent experiments is shown at the bottom. Error bars represent standard deviations. The translational yield of hfq and of hfqΔ30 mRNAs is indicated by gray and white bars, respectively. The translational yield obtained with either mRNA in the absence of Hfq was set to 1. (B) Hydroxyl radical footprints of hfq96 mRNA in the presence of Hfq. Samples containing 5′-end labeled hfq96 mRNA were incubated in the absence (lane 1) or in the presence of Hfq (lanes 2–4) and subjected to hydroxyl radical cleavage. Hfq was added in fourfold (lane 2), eightfold (lane 3), and 16-fold (lane 4) molar excess (Hfq₆ excess) over hfq96 mRNA. Lane 5, untreated hfq96 mRNA (5 nM) was incubated with Hfq (80 nM Hfq₆). Lane 6, RNase T1 cleavage (G-specific cleavage) of hfq96 mRNA. The region protected from hydroxyl-radical cleavage by Hfq (site B) is marked by a bar. Nucleotide positions in the hfq mRNA sequence are shown at the right. (C) Relative translational efficiency (see Materials and Methods) of the hfq101-lacZ mRNA in a hfq⁺ and an hfq⁻ genetic background. The expression of the plasmid-encoded hfq101-lacZ gene was induced in the hfq⁻ strain AM111F′ and in the wild-type strain MC4100F′ as described in Materials and Methods. The averaged β-galactosidase values normalized to mRNA levels obtained in the hfq⁻ strain (control) was set to 1 (white bar). The value obtained with the hfq⁺ strain (gray bar) was normalized to the control. The experiment was performed in duplicate. The error bars represent standard deviations.