Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli

Abstract

Escherichia coli possesses a unique RNase activity that cleaves stop codons in the ribosomal aminoacyl-tRNA binding site (A-site) during inefficient translation termination. This A-site mRNA cleavage allows recycling of arrested ribosomes by facilitating recruitment of the tmRNA*SmpB ribosome rescue system. To test whether A-site nuclease activity also cleaves sense codons, we induced ribosome pausing at each of the six arginine codons using three strategies; rare codon usage, arginine starvation, and inactivation of arginine tRNAs with colicin D. In each instance, ribosome pausing induced mRNA cleavage within the target arginine codons, and resulted in tmRNA-mediated SsrA-peptide tagging of the nascent polypeptide. A-site mRNA cleavage did not require the stringent factor ppGpp, or bacterial toxins such as RelE, which mediates a similar nuclease activity. However, the efficiency of A-site cleavage was modulated by the identity of the two codons immediately upstream (5' side) of the A-site codon. Starvation for histidine and tryptophan also induced A-site cleavage at histidine and tryptophan codons, respectively. Thus, A-site mRNA cleavage is a general response to ribosome pausing, capable of cleaving a variety of sense and stop codons. The induction of A-site cleavage during amino acid starvation suggests this nuclease activity may help to regulate protein synthesis during nutritional stress.

Figure 1. A-site mRNA cleavage of rare Arg codons

(a) The model flag-trxA(PPRR) mRNA is depicted with FLAG-encoding region and oligonucleotide probe binding sites indicated. Arg-84 of all flag-trxA(PPRR) constructs was changed to Lys as indicated (R84K). Flag-TrxA-PPRR residues Ala-116 to Arg-121 are shown along with the encoding mRNA sequence and the complementary sequence of the S1 nuclease protection probe. Arg-120 and Arg-121 were coded as tandem AGG, AGA or CGG codons as indicated. The 3′ end of the truncated in vitro transcript used in Northern blot and S1 protection analysis is shown. Arrows indicate the positions of BsaJI, NlaIV and BamHI endonuclease cleavages in the S1 protection probe used to generate gel migration standards. (b) Northern blot analyses of RNA from ΔtmRNA cells. Samples from cells expressing mRNA containing tandem AGG, AGA or CGG codons (labeled accordingly) were probed with a radiolabeled oligonucleotide probe specific for the ribosome binding site (RBS) of mRNA as depicted in (a). Lanes labeled 5, 4 and 3 indicate the samples came from ΔtmRNA cells overproducing tRNA₅^Arg (decodes AGG), tRNA₄^Arg (decodes AGA and AGG) and tRNA₃^Arg (decodes CGG), respectively ; . The position of truncated flag-trxA(PPRR)_AGG mRNA is indicated by the arrow. The control in vitro transcript was truncated after the first AGG codon as shown in (a). Northern analysis of tRNA^Arg species was performed using labeled oligonucleotide probes specific for each tRNA. (c) S1 nuclease protection of truncated flag-trxA(PPRR)_AGG transcripts from ΔtmRNA cells was similar to that of the truncated control in vitro transcript, consistent with cleavage in and around the AGG codons. These protections were not seen in samples taken from tmRNA⁺ cells or ΔtmRNA cells overproducing tRNA₄^Arg or tRNA₅^Arg. No S1 protection was observed if the flag-trxA(PPRR)_AGG mRNA was not induced with IPTG. The relative positions of BamHI, BsaJI and NlaIV digestion sites in the S1 probe with respect to the tandem AGG codons are shown in (a).

Figure 2. Effect of nascent peptide on mRNA cleavage

(a) Northern blot analyses of RNA isolated from ΔtmRNA cells. Translation of each mRNA leads to ribosome pausing at tandem rare AGG Arg codons in the context of Pro-Pro (PPRR), Leu-Ala (LARR), Ser-His (SHRR), Asp-Thr (DTRR) or Lys-Lys (KKRR) nascent peptides. Overproduction of tRNA₅^Arg suppressed the accumulation of truncated mRNA in each case. The position of truncated flag-trxA(PPRR)_AGG mRNA (as described in Fig. 1a) is indicated by the arrow. Larger truncated transcripts were observed in cells expressing flag-trxA(LARR)_AGG and flag-trxA(KKRR)_AGG messages. (b) S1 nuclease protection of flag-trxA(KKRR)_AGG message from ΔtmRNA cells showed prominent cleavages 12 – 17 nucleotides downstream of the first AGG codon. These protections were not seen ΔtmRNA cells overproducing tRNA₅^Arg. No S1 protection was observed if the flag-trxA(KKRR)_AGG mRNA was not induced with IPTG. (c) Flag-TrxA-KKRR was expressed in tmRNA(His₆) cells and purified by Ni²⁺ affinity chromatography. Mass spectrometry detected two species corresponding to the SsrA(His₆) tag added after Lys-119 (calculated mass, 14,423.3 Da) and Arg-120 (calculated mass, 14,579.5). The same SsrA-peptide tagging sites were identified in each protein variant (data not shown).

Figure 3. Analysis of translational frameshifting at rare Arg codons

(a) Flag-TrxA protein variants were produced in ΔtmRNA cells and total protein analyzed by Western blot using a monoclonal antibody specific for the N-terminal FLAG epitope. The +1 frameshift products and full-length proteins are indicated. Overproduction of tRNA₅^Arg suppressed +1 frameshifting in each construct (+ptRNA₅^Arg, and data not shown). (b) The percentages of +1 frameshifting were determined from anti-FLAG fluorescent Western blot data using LI-COR^® Odyssey software. The reported values are the average ± standard deviation determined from five independently prepared total protein samples. (c) The percentages of A-site cleaved and 3′-boundary cleaved products relative to full-length mRNA were determined from Northern blot phosphorimager data using the Quantity One (BioRad) software package. Reported values are the average ± standard deviation determined from five independently prepared total RNA samples.

Figure 4. Arginine starvation induces mRNA cleavage and tmRNA-mediated peptide tagging activities

(a) Northern blot analyses of RNA isolated from ΔtmRNA cells that were arginine-fed (+) or starved for arginine (−). For each codon construct, truncated mRNA accumulated under arginine starvation conditions. The position of truncated mRNA indicated by the arrow corresponds to a control in vitro transcript of flag-trxA(PPRR)_CGU that was truncated after the first CGU codon. RNA was resolved at low pH on acid-urea gels for Northern analysis of tRNA₂^Arg. Under these conditions, aminoacylated tRNA₂^Arg was resolved from deacylated tRNA₂^Arg. The sample loaded in the in vitro lane contained 0.2 pmole of truncated in vitro transcript mixed with 10 μg of total RNA that had been alkali-treated to deacylate tRNA. (b) Flag-TrxA-PPRR was produced in tmRNA(DD) cells under arginine-fed (+) or arginine-starved (−) conditions. Total protein was analyzed by SDS-PAGE followed by Coomassie blue staining, and by Western blot analysis using antibodies specific for the SsrA(DD) peptide tag. SsrA(DD) peptide tagging was only observed in response to arginine starvation. (c) Flag-TrxA-PPRR from the flag-trxA(PPRR)_CGU construct was expressed in tmRNA(His₆) cells under arginine starvation conditions and purified by Ni²⁺ affinity chromatography. Mass spectrometry detected two species corresponding to SsrA(His₆) tag addition after Pro-119 (calculated mass, 14,361.2 Da) and Arg-120 (calculated mass, 14,517.4). Essentially identical results were obtained with Flag-TrxA-PPRR protein produced from the CGA and CGC coded constructs (data not shown).

Figure 5. S1 nuclease mapping of arginine starvation and colicin D-induced mRNA cleavages

(a) The trxA(PPRR)_CGU mRNA is depicted with FLAG encoding region and oligonucleotide probe binding sites indicated. Residues Ala-116 to Arg-121 of Flag-TrxA-PPRR are shown along with the encoding mRNA sequence and the complementary sequence of the S1 nuclease protection probe. Arg-120 and Arg-121 were coded as tandem CGU codons, and Arg-84 was changed to Lys as indicated (R84K). The truncated in vitro transcript used in Northern blot (Fig. 3a) and S1 protection analysis is shown, indicating the position of the 3′ terminus. Arrows indicate the positions of HgaI, NlaIV and BamHI endonuclease cleavages in the S1 probe used to generate gel migration standards. (b) The main S1 nuclease protections seen in ΔtmRNA cells subjected to arginine starvation mapped to the tandem CGU codons. Similar protections were seen in arginine-starved ΔtmRNA cells that were also deleted for RelE and related toxins (Δtoxins), and ΔtmRNA cells lacking ppGpp (ppGpp⁰). These protections were not seen in samples taken from tmRNA⁺ cells. In ΔtmRNA cells, colicin D treatment resulted in cleavages in the CGU codons and at sites 12 nucleotides downstream of second CGU codon, whereas only the downstream cleavages were detected in tmRNA⁺ cells. No S1 protection was observed in uninduced cells (no IPTG). The relative positions of HgaI, NlaIV and BamHI digestion sites in the S1 probe with respect to the tandem CGU codons are shown in (a).

Figure 6. Colicin D treatment induces mRNA cleavage and tmRNA-mediated peptide tagging activities

(a) Northern blot analyses of RNA isolated from cells treated with colicin D. For each codon construct, colicin D treatment produced truncated mRNA not observed in the untreated sample (no colicin D). Two distinct truncated messages were detected in ΔtmRNA cells, whereas only one accumulated in tmRNA⁺ cells. The position indicated by the arrow corresponds to a control in vitro transcript of trxA(PPRR)_CGU truncated after the first CGU codon (in vitro). Northern analysis for tRNA₂^Arg showed a substantial fraction of the tRNA was cleaved in response to colicin D treatment. The sample loaded in the in vitro lane contained 0.2 pmole of truncated in vitro transcript mixed with 10 μg of total RNA isolated from cells that had not been incubated with IPTG or colicin D. (b) Flag-TrxA-PPRR was produced in tmRNA(DD) cells either treated with colicin D (+) or not (−). Total protein was analyzed by SDS-PAGE followed by Coomassie blue staining, and by Western blot analysis using antibodies specific for the SsrA(DD) peptide tag. SsrA(DD) peptide tag addition was only observed in samples taken from colicin D treated cells.

Figure 7. Histidine and tryptophan starvation induces mRNA cleavage and tmRNA-mediated peptide tagging activities

(a) Northern blot analyses of RNA isolated from ΔtmRNA cells that were fed (+) or starved (−) for histidine or tryptophan. Truncated mRNA from the CAC and CAU containing constructs was only produced during histidine starvation, and tryptophan starvation induced cleavage of the UGG containing mRNA. The position of truncated mRNA indicated by the arrow corresponds to a control in vitro transcript of flag-trxA(PPHHLA)_CAU that was truncated after the first CAU codon. RNA was resolved at low pH on acid-urea gels for Northern analysis of tRNA^His and tRNA^Trp. Under these electrophoresis conditions, aminoacylated tRNAs were resolved from deacylated tRNAs. The sample loaded in the in vitro lane contained 0.2 pmole of truncated in vitro transcript mixed with 10 μg of total RNA that had been alkali-treated to deacylate tRNA. (b) Flag-TrxA-PPHHLA and Flag-TrxA-PPWWLA proteins were produced in tmRNA(DD) cells under amino acid-fed (+) or amino acid-starved (−) conditions. Total protein was analyzed by SDS-PAGE followed by Coomassie blue staining, and by Western blot analysis using antibodies specific for the SsrA(DD) peptide tag. SsrA(DD) peptide tag addition was only observed during amino acid starvation. (c) Flag-TrxA-PPWWLA was expressed in tmRNA(His₆) cells under tryptophan starvation conditions and purified by Ni²⁺ affinity chromatography. Mass spectrometry detected two species corresponding to SsrA(His₆) peptide tag addition after Pro-119 (calculated mass, 14,283.1 Da) and Trp-120 (calculated mass, 14,469.3 Da).

Figure 8. S1 nuclease mapping of tryptophan starvation-induced mRNA cleavages

(a) The trxA(PPWWLA) mRNA is depicted with FLAG coding region and oligonucleotide probe binding sites indicated. Trp-39 and Trp-42 were changed to Phe residues as indicated. Residues Ala-116 to Ala-123 of Flag-TrxA-PPWWLA are shown along with the encoding mRNA sequence and the complementary sequence of the S1 nuclease probe. The truncated in vitro transcript used in Northern blot and S1 protection analysis is shown, indicating the position of the 3′-terminus. Arrows indicate the positions of HgaI, NlaIV and BamHI endonuclease cleavages in the S1 probe used to generate gel migration standards. (b) S1 nuclease protections detected in ΔtmRNA cells subjected to tryptophan starvation mapped to the tandem UGG codons. These protections were not seen in tmRNA⁺ cells or ΔtmRNA cells that were fed tryptophan. No S1 protection was observed in uninduced cells (no IPTG). The relative positions of HgaI, NlaIV and BamHI digestion sites in the S1 probe with respect to the tandem UGG codons are shown in (a).