The RNA i-motif

International audienc

Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

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