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.
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106-9610, USA.
Laura E
LE
Holberger
Christopher S
CS
Hayes
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008156:Luciferases / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010303:Paromomycin / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013307:Streptomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C034212:ribosomal protein S12; C031798:ribosomal protein S4; C430328:small protein B; C096222:tmRNA; D010303:Paromomycin; D008156:Luciferases; D013307:Streptomycin