Programmed Ribosomal Frameshifting Generates a Copper Transporter and a Copper Chaperone from the Same Gene

Abstract

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo.

Keywords: copA; nascent peptide; pseudoknot; recoding; ribosome profiling; slippery sequence; translation regulation.

Figure 1. The short polypeptide CopA(Z) is translated in the cell from the E. coli copA gene

(A) Ribosome profiling reveals an abrupt drop in ribosome density around the 70^th codon of the copA gene (black arrow). The boundaries of the two metal binding domains MBD1 and MBD2 of the CopA transporter protein encoded in the copA gene are indicated; MBD1 is colored grey. The profiling data are from the control cells in (Kannan et al., 2014). (B) Two left panels: Coomassie-stained 15% SDS gel showing low-molecular weight proteins in the control and IPTG-induced ΔcopA E. coli cells carrying the pCopA plasmid which encodes N-terminally His₆-tagged CopA. Right: silver-stained 4-20% SDS gel showing the N-terminally His₆-tagged short protein expressed in the IPTG-induced cells and purified by Ni²⁺-affinity chromatography. The short protein with the electrophoretic mobility approximately corresponding to the His₆-tagged MBD1 of CopA, that we named CopA(Z), is indicated by arrowheads. (C) Top-down mass-spectrometric analysis of the purified His₆-tagged CopA(Z). The deconvoluted monoisotopic mass of 9323.61 Da is consistent with the predicted mass of CopA(Z) (9337.60 Da), where the C-terminal Ala, encoded in the 70^th codon of copA, is replaced with Gly. The spectrum shows multiply charged species of the intact protein; for reference, the peak corresponding to the charge 10+ is indicated by an arrow for reference. The amino acid sequence of His₆-tagged CopA(Z) is shown below and the C-terminal Gly is boxed.

Figure 2. Production of CopA(Z) depends on the integrity of the slippery sequence present in the copA gene

(A) The slippery sequence CCCAAAG present in the copA gene at the end of the MBD1-coding segment. Slippage of the ribosome to the -1 frame would cause the ribosome to incorporate Gly-70 after Lys-69 and terminate translation at the following stop codon, generating the CopA(Z) protein. (B) Immunoblot of the lysates of E. coli cells carrying either the pCopA plasmid containing the wt copA gene, or pCopA-mSS in which the slippery sequence was mutated to prevent -1 PRF. The CopA (grey arrowhead) and CopA(Z) (black arrowhead) proteins were detected using anti-His₆-tag antibodies. Proteins were fractionated in a 4-20% SDS gel. The SS-disrupting mutations (which do not change the sequence of the encoded protein) are underlined. (C) In vitro transcription-translation of a DNA template containing the first 94 codons of copA, followed by an engineered stop codon. The 16.5% Tris-Tricine SDS gel shows the [³⁵S]-labeled products corresponding to the complete 94 amino acid-long protein encoded in the 0 frame (grey arrowhead) or the 70 amino acid-long CopA(Z) produced via -1 PRF (black arrowhead). Disruption of SS in the template mSS by two synonymous mutations (shown in panel B) prevents production of CopA(Z) in vitro. See also Figure S2.

Figure 3. mRNA structure downstream of SS promotes efficient -1 PRF in the copA gene

(A) The predicted structure of the pseudoknot in the copA mRNA downstream from the SS. The slippery site is shaded in gray, the -1 frame stop codon is boxed, and stems S1 and S2 of the pseudoknot are indicated. (B) Predicted secondary structure of the mRNA segments downstream of the SS in the 3′-truncated copA templates. The boundaries of the CopA(Z) coding segment is indicated and the location of SS is marked by an arrow. (C) SDS-gel analysis of the [³⁵S]-labeled protein products expressed in vitro from the 3′-truncated copA templates shown in (B). Grey arrowheads indicate the bands corresponding to the full-sized 0 frame product; black arrowheads indicate CopA(Z) generated via -1 PRF. (D) Quantification of the -1 PRF efficiency in the different 3′-truncated copA templates from the relative intensity of the bands indicated in (C). Error bars show standard deviation from the mean in 3 independent experiments. See also Figure S3.

Figure 4. The CopA nascent peptide contributes to the high efficiency of -1 PRF

(A) Cartoon representation of the ribosomes translating through the copA SS sequence of the wt template (copA_1-312) or of the NP1 and NP2 mutants. The sequence of the wt CopA nascent chain is shown in grey and the mutant peptide sequence is shown in black. The nucleotide and amino acid sequences of the templates are show in detail Figure S1. (B) Gel electrophoretic analysis of the [³⁵S]-labeled truncated CopA encoded in the 0 frame of the copA_1-312 wt or mutant templates (gray arrowheads) and CopA(Z) generated via -1 PRF (black arrowheads). The reference templates (M) encode the marker CopA(Z) proteins whose sequences matched those generated from the mutant templates via -1 PRF. The sequences of the reference templates are shown in Figure S1. (C) Quantification of the -1 PRF efficiency in the different copA templates from the relative intensity of the bands shown in (B). Error bars show deviation from the mean in 3 independent experiments. (D) The structures of the dual luciferase reporters containing unaltered (wt) or mutant (NP2) CopA segments. The construct carrying the copA sequence with the mutated SS (mSS) was used as a negative control. (E) Efficiency of the in vivo -1 PRF in the dual luciferase reporters shown in (D). Error bars show standard deviation from the mean in 3 independent experiments. See also Figure S1.

Figure 5. Production of the diffusible metal chaperone CopA(Z) helps survival during copper stress

Co-growth competition of the wt and the mSS mutant E. coli cells. Production of CopA(Z) in the mSS mutants was prevented by introduction of two synonymous mutations which disrupted the SS in the chromosomal copA gene. Equal numbers of wt and mutant cells were mixed and passaged in liquid culture in the absence or presence of toxic (4 mM) concentration of CuSO₄. Sequencing chromatograms of the copA segment PCR-amplified from the genomic DNA isolated from the co-cultures used to assess the ratio of wt and mSS cells at the (A) onset of the experiment, after (B) 30 generations or (C) 50 generations of growth in the presence of CuSO₄. The wt copA sequence contains C at position 201 and A at position 204 within the CCCAAAG slippery sequence; these residues were mutated to T in the mSS mutant. The arrows indicate the sequencing chromatograms peaks corresponding to these nucleotides. (D) The ratios between wt and mSS cells in the co-culture were computed from the height of the chromatogram peaks. Error bars show standard deviation from the mean in 3 independent experiments.

Figure 6. -1 PRF element within the human copper transporter ATP7B gene

(A) The location of the heptameric CCCAAAG SS of the human ATP7B gene. Translation in 0 frame generates the full-size ATP7B copper transporter; slippage of the ribosome into -1 frame (underlined codons) would cause termination of translation at the 240^th codon (UAA) and result in production of a truncated protein consisting of the first two MBDs of ATP7B. (B) The ATP7B mRNA segment downstream of the slippery sequence could fold into a PK. The SS is underlined and the -1 frame stop codon is boxed. (C) The dual luciferase reporter constructs used to test efficiency of -1 PRF directed by the ATP7B SS. The minimal ATP7B sequence including the SS and the putative PK structure was inserted between the Rluc (0 frame) and the Fluc (-1 frame) genes. The premature termination codon (PTC) control construct carried a stop codon at the end of the Rluc gene. The out of frame (OOF) control construct contained a stop codon at the beginning of the -1 frame Fluc ORF. (D) The -1 PRF directed by the HIV-1 PRF signal in comparison with the ATP7B PRF element within the constructs shown in panel (C) expressed in HEK293T mammalian cells. The errors bars represent standard deviation from the mean based on at least 6 independent replicates. See also Figure S4.