Holberger LE, Hayes CS
Ribosomal protein S12 and aminoglycoside antibiotics modulate A-site mRNA cleavage and transfer-messenger RNA (tmRNA) activity in Escherichia coli. [JOURNAL ARTICLE]
J Biol Chem 2009 Sep 23.
Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A site. A-site mRNA cleavage is thought to facilitate 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 to 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.
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