Ribosomal protein S12 and aminoglycoside antibiotics modulate A-site mRNA cleavage and transfer-messenger RNA activity in Escherichia coli

Abstract

Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA).SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL(+) cells. Additionally, tmRNA.SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA.SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA.SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA.SmpB activity. We propose that tmRNA.SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants.

FIGURE 1.

Aminoglycoside binding sites in the ribosomal A-site. The decoding center of the 30 S subunit is depicted with helix 44 (h44) of 16 S rRNA and ribosomal protein S12. 16 S rRNA residues G⁵³⁰, A¹⁴⁹², and A¹⁴⁹³ are indicated along with the ribosome binding sites of streptomycin (red) and paromomycin (green). Mutations that change S12 residues Lys⁴² and Pro⁹⁰ are able to confer both streptomycin-resistant and streptomycin-dependent phenotypes. These data were taken from PDB accession number 1FJG (19) and rendered with PyMol.

FIGURE 2.

Streptomycin inhibits A-site mRNA cleavage during inefficient translation termination. A, FLAG-λN expression constructs. The generalized flag-λN transcript is shown schematically along with the Northern probe-binding site. The 3′-coding sequences of the flag-λN(PP), flag-λN(LA), and flag-λN(UAA→Gln) transcripts are shown. The UAA→Gln mutation changes the UAA stop codon to a glutamine codon, mimicking stop codon read-through. The boxed P and A indicate the positions of the ribosomal P- and A-sites during translation termination. B, Northern blot analysis of flag-λN(PP) mRNA. The flag-λN(PP) transcript was expressed in tmRNA⁺ and ΔtmRNA cells as indicated. ΔtmRNA cells were treated with increasing concentrations of streptomycin for 15 min, total RNA was isolated, and the effects on A-site cleavage were assessed by Northern blot. The 3′-end of the truncated transcript was mapped to the stop codon by S1 nuclease protection analysis (data not shown). The positions of full-length and A-site-truncated transcripts are indicated. C, Western blot analysis of protein synthesis in streptomycin-treated cells. Expression of FLAG-λN(PP) was induced in ΔtmRNA cells concomitantly with the addition of 7.5 μm streptomycin, and samples were taken at the indicated times for Western blot using FLAG-specific antibodies. Streptomycin induced the production of an alternative translation product that co-migrated with protein expressed from flag-λN(UAA→Gln), consistent with stop codon read-through. Streptomycin did not induce stop codon read-through during FLAG-λN(LA) synthesis.

FIGURE 3.

Paromomycin inhibits A-site mRNA cleavage during inefficient translation termination. A, λN-FLAG-His₆ expression constructs. The generalized λN-flag-His₆ transcript is shown schematically along with the Northern probe-binding site. The 3′-coding sequences of λN-flag-His₆(PP) and λN-flag-His₆(LA) messages are shown. The boxed P and A indicate the positions of the ribosomal P- and A-sites during translation termination. B, Northern blot analysis of λN-flag-His₆(PP) mRNA. The λN-flag-His₆(PP) message was expressed in tmRNA⁺ and ΔtmRNA cells as indicated. ΔtmRNA cells were treated with increasing concentrations of paromomycin, and the effects on A-site cleavage were assessed by Northern blot. The 3′-end of the truncated transcript was mapped to the stop codon by S1 nuclease protection analysis (data not shown). The positions of full-length and A-site-truncated transcripts are indicated. C, Western blot analysis of protein synthesis in paromomycin-treated cells. Expression of λN-FLAG-His₆(PP) was induced in ΔtmRNA cells concomitantly with the addition of paromomycin at various concentrations. Cells were collected after 20 min, and total urea-soluble protein was isolated and subjected to Western blot analysis using FLAG-specific antibodies. The rate of λN-FLAG-His₆(PP) synthesis in ΔtmRNA cells treated with 20 μm paromomycin was also compared with untreated cells. Finally, paromomycin induced stop codon read-through during the synthesis of λN-FLAG-His₆(PP) but not λN-FLAG-His₆(LA).

FIGURE 4.

SsrA peptide tagging during aminoglycoside treatment. A, mass spectrometry of SsrA(His₆)-tagged FLAG-λN(PP). FLAG-λN(PP) was expressed in tmRNA(His₆) cells, and SsrA(His₆)-tagged protein was purified by Ni²⁺ affinity chromatography for mass spectrometry. The major species had a mass of 13,365 Da, corresponding to SsrA(His₆) tag addition after the C-terminal Pro residue (calculated mass, 13,364.9 Da). B, Western blot analysis of SsrA(DD) peptide tagging during aminoglycoside treatment. FLAG-λN(PP) and λN-FLAG-His₆(PP) were expressed in tmRNA(DD) cells treated with increasing concentrations of aminoglycosides for 20 min. Total urea-soluble protein was analyzed by Western blot using antibodies specific for the FLAG and SsrA(DD) epitopes. Individual fluorescence channels and the merged signals are presented.

FIGURE 5.

tmRNA·SmpB-mediated ribosome rescue from nonstop mRNA during aminoglycoside treatment. A, schematic of tmRNA-mediated and tmRNA-independent ribosome recycling. The λN-flag-His₆(trpL) nonstop message is depicted, and the positions of the encoded flag and His₆ peptide epitopes are indicated. The λN-flag-His₆ open reading frame was fused to the intrinsic transcription terminator from the E. coli trp leader to create a transcript lacking in-frame stop codons. Ribosomes arrested on nonstop messages are efficiently rescued by the tmRNA·SmpB system. In ΔtmRNA cells, the nascent chains are released from these stalled ribosomes by an uncharacterized pathway, and untagged protein accumulates. B, Western blot analysis of SsrA(DD) peptide tagging during nonstop mRNA expression. The λN-flag-His₆(trpL) transcript was expressed in ΔtmRNA and tmRNA(DD) cells as indicated. tmRNA(DD) cells were treated with increasing concentrations of streptomycin and paromomycin for 20 min. Total urea-soluble protein was analyzed by Western blot using antibodies specific for the FLAG and SsrA(DD) epitopes. Individual fluorescence channels and the merged signals are presented.

FIGURE 6.

Northern analysis of A-site mRNA cleavage in streptomycin-resistant rspL mutants. The flag-λN(PP) transcript was expressed in ΔtmRNA cells containing rpsL mutations that encode the indicated streptomycin-resistant S12 variants. Total RNA was isolated from cells grown in the absence and presence of streptomycin (50 μm) as indicated. Samples from tmRNA⁺ and ΔtmRNA cells containing wild-type S12 (rpsL⁺) were included in each blot as a reference control for A-site cleavage. The rpsL(P90K) and rpsL(P90R) mutants are streptomycin-dependent and therefore were not tested in the absence of streptomycin. Similarly, the rpsD mutations confer streptomycin sensitivity to the parental rpsL(P90K) strain, and therefore these mutants were not tested with streptomycin. The positions of full-length and A-site-truncated transcripts are indicated. The percentage of A-site-truncated transcripts, with respect to total transcript (full-length + truncated), was determined from phosphorimaging data as described under “Experimental Procedures.”

FIGURE 7.

Quantification of SsrA(DD) peptide tagging in streptomycin-resistant rpsL mutants. The efficiency of SsrA(DD) peptide tagging during inefficient translation termination was determined using λN-FLAG-His₆(PP) as the reporter protein. λN-FLAG-His₆(PP) was synthesized in rpsL mutants expressing tmRNA(DD) in the absence or presence of streptomycin (50 μm) as indicated. Total λN-FLAG-His₆(PP) protein was purified by Ni²⁺ affinity chromatography, and the SsrA(DD)-tagged and -untagged chains were resolved by SDS-PAGE and quantified using LI-COR® Odyssey software. The percentage of tagged chains in each background is reported as the average ± S.E. from three independently conducted experiments.

FIGURE 8.

Ribosome rescue activity in streptomycin-resistant rpsL mutants. The efficiency of tmRNA·SmpB-mediated ribosome rescue from nonstop mRNA was assessed by SsrA(DD) peptide tagging. λN-FLAG-His₆ was synthesized from a nonstop message (shown in Fig. 5A) in tmRNA(DD) cells containing the indicated rpsL mutations. Cells were grown in the absence or presence of streptomycin (50 μm) as indicated. Total λN-FLAG-His₆(PP) protein was purified by Ni²⁺ affinity chromatography, and the SsrA(DD)-tagged and untagged chains were resolved by SDS-PAGE, followed by Coomassie Blue staining.

FIGURE 9.

Decoding fidelity of streptomycin-resistant cells. The pAD8 plasmid encoding a Renilla-firefly luciferase fusion interrupted by a UGA stop codon at position 417 of firefly luciferase was introduced into ΔtmRNA cells containing the indicated rpsL mutations. Increased stop codon read-through results in a higher ratio of firefly to Renilla luciferase activity (F-Luc/R-Luc). Firefly luciferase/R-Luc ratios were determined from lysates of cells grown in the absence and presence of streptomycin (50 μm) as indicated. Reported values are the average ± S.E. for at least three independently conducted experiments.