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.
Highlights
Translational pausing in Escherichia coli elicits a unique RNase activity that cleaves the A-site codon within paused ribosomes [1, 2]
Aminoglycoside Antibiotics Inhibit A-site mRNA Cleavage— We examined the effect of streptomycin on A-site mRNA cleavage using a plasmid-borne construct that expresses the N-terminal domain of phage cI repressor containing a C-terminal Pro-Pro sequence (Fig. 2A)
To determine whether aminoglycosides inhibit SsrA peptide tagging independent of stop codon read-through, we examined tmRNA1⁄7SmpB activity with ribosomes stalled on nonstop mRNA
Summary
X90 ⌬ssrA CH165 rpsL(K42R), StrR CH165 rpsL(K42N), StrR CH165 rpsL(K42T), StrR CH22 rpsL(K42A), CmR, StrR CH165 rpsL(K42C), StrR CH165 rpsL(K42S), StrR CH165 rpsL(K42V), StrR CH165 rpsL(K42Y), StrR CH165 rpsL(P90F), StrR CH165 rpsL(P90Y), StrR CH165 rpsL(P90H), StrR CH165 rpsL(P90N), StrR CH165 rpsL(P90Q), StrP CH165 rpsL(P90R), StrD CH22 rpsL(P90K), CmR, StrD CH22 rpsL(P90K) rpsD-1(Q53K), CmR, StrS CH22 rpsL(P90K) rpsD-2(N85Y), CmR, StrS CH22 rpsL(P90K) rpsD-3(Y203amb), CmR, StrS CH22 rpsL(P90K) rpsD-4(A192-TLTNT), CmR, StrS. Ref. 22 Ref. 1 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study. Plasmids pTrc99A pFG501 pFLAG-N(PP) pFLAG-N(LA) pPW500 pN-FLAG-His6(PP) pN-FLAG-His6(LA) pKW1. Isopropyl 1-thio--D-galactopyranoside-inducible expression vector, AmpR FLAG-encoding derivative of pTrc99A, AmpR pFG501 derivative for inducible expression of FLAG-N(PP), AmpR pFG501 derivative for inducible expression of FLAG-N(LA), AmpR Expresses N-FLAG-His from trp leader terminated nonstop mRNA, AmpR pTrc99A derivative expressing N-FLAG-His6(PP), AmpR pTrc99A derivative expressing N-FLAG-His6(LA), AmpR pACYC184 derived plasmid, TetR pKW1-derived plasmid expressing wild-type tmRNA, TetR pKW1-derived plasmid expressing tmRNA(DD), TetR pKW1-derived plasmid expressing tmRNA(His6), TetR Encodes Renilla-firefly luciferase fusion interrupted by UGA stop codon, AmpR
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