Inhibition of Transfer Messenger RNA Aminoacylation and trans-Translation by Aminoglycoside Antibiotics

Sophie Corvaisier,Valérie Bordeau,Brice Felden

doi:10.1074/jbc.m212830200

Abstract

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.

Highlights

In bacteria, transfer messenger RNA1 known alternatively as SsrA RNA or 10Sa RNA, rescues stalled ribosomes and contribute to the degradation of incompletely synthesized peptides
Inhibition of Transfer messenger RNA (tmRNA) Aminoacylation by Aminoglycoside Antibiotics—Aminoacylation experiments with purified E. coli alanyl-tRNA synthetase were performed on two RNAs purified in vivo: tmRNA from E. coli (363 nucleotides; Fig. 1A) and tmRNA-TLD (61 nucleotides; Fig. 1B), a shorter RNA recapitulating the tRNA-like domain of tmRNA. tmRNA-TLD is capable of being aminoacylated with alanine in vitro
Functional and structural evidence is provided to demonstrate that aminoglycoside antibiotics interact in vitro with tmRNA from E. coli and modify its conformation in solution, resulting in its inability to be efficiently aminoacylated with alanine by alanyl-tRNA synthetase. tmRNA aminoacylation was most strongly inhibited by neomycin B and by paromomycin followed by, in descending order, gentamicin, amikacin, kanamycin A, and tobramycin (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

Enzymes and RNAs—Alkaline phosphatase and T4 polynucleotide kinase are from New England Biolabs (Beverly, MA). Kinetic parameters (Km and Vmax) in the presence and absence of aminoglycosides were performed under steady-state conditions of enzyme (50 –330 nM AlaRS) and substrate concentrations of tmRNA (0.5–3.4 ␮M) or tmRNA-TLD (0.5– 6 ␮M) and determined from Lineweaver-Burk plots These experimental conditions were applied for tmRNA aminoacylation in the presence of all tRNAs from E. coli. Chemical Footprints—Labeling at the 5Ј ends of tmRNA and tmRNA-TLD were performed with [␥-32P]ATP and phage T4 polynucleotide kinase on RNA dephosphorylated previously with alkaline phosphatase. Labeling at their 3Ј ends was carried out by ligation of [␥-32P]pCp using T4 RNA ligase. Because the overall conformation of tmRNA changes in the presence of both aminoglycosides, the conformation of tmRNA in the presence of the aminoglycosides might be less sensitive to the action of the structural probe

RESULTS

35 Ϯ 15 500 Ϯ 100 700 Ϯ 100 900 Ϯ 100

23 Ϯ 5 26 Ϯ 5

DISCUSSION