Erratum: Aminoglycoside activity observed on single pre-translocation ribosome complexes

Abstract

Aminoglycoside-class antibiotics bind directly to ribosomal RNA, imparting pleiotropic effects on ribosome function. Despite in-depth structural investigations of aminoglycoside–RNA oligonucleotide and aminoglycoside-ribosome interactions, mechanisms explaining the unique ribosome inhibition profiles of chemically similar aminoglycosides remain elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) methods, we show that high-affinity aminoglycoside binding to the conserved decoding site region of the functional pre-translocation ribosome complex specifically remodels the nature of intrinsic dynamic processes within the particle. The extents of these effects, which are distinct for each member of the aminoglycoside class, strongly correlate with their inhibition of EF-G–catalyzed translocation. Neomycin, a 4,5-linked aminoglycoside, binds with lower affinity to one or more secondary binding sites, mediating distinct structural and dynamic perturbations that further enhance translocation inhibition. These new insights help explain why closely related aminoglycosides elicit pleiotropic translation activities and demonstrate the potential utility of smFRET as a tool for dissecting the mechanisms of antibiotic action.

Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex

“…This demonstration of the metastable nature of the mammalian PRE complex suggests that cycloheximide binding specifically promotes classical configurations to inhibit translocation by directly interfering with P/E hybrid state formation. These observations are reminiscent of the action of unrelated aminoglycoside antibiotics on the bacterial PRE complex, where classical state stabilization was shown to directly correlate with translocation inhibition (Feldman et al, 2010). …”

High-resolution structure of the Escherichia coli ribosome

“…smFRET experiments were performed using a prism-based total internal reflection (TIR) microscope 30 , 62 at 25 °C in Tris-polymix buffer 61 with an oxygen scavenging system 63 , triplet-state quenching compounds 64 (1 mM cyclooctatetraene, 1 mM nitrobenzyl alcohol, 1 mM Trolox) and 2.5 to 30 mM Mg 2+ . After surface immobilization of initiation complexes programmed with biotinylated mRNA to PEG-passivated, streptavidin-coated quartz microfluidic chambers 30 , 62 , pre-translocation complexes were prepared by incubating with either unlabeled ternary complex of EF-Tu(GTP)Phe-tRNA Phe (for the intersubunit rotation assay) or EF-Tu(GTP)Phe-tRNA Phe (Cy5-acp 3 U47) (for the tRNA hybrid state assay) for 2 min in Tris-polymix buffer, as previously described 4 . Cy3 fluorophores on either uS13 or P-site tRNA were illuminated with a 532 nm diode-pumped solid-state laser (Opus, LaserQuantum).…”

“…smFRET data were acquired at 25 ms integration time and analyzed using analytical software implemented in MATLAB (MathWorks). FRET trajectories were idealized using the segmental k-means algorithm 65 , as previously described 4 , 61 . Experiments were performed using three technical replicates, i.e.…”

“…In the bacterium Escherichia coli , ribosomes harbor large numbers of different cations, including potassium, magnesium, and polyamines. These ions influence ribosome dynamics, including intersubunit rotation 2 as well as the mechanisms of mRNA decoding 3 and translocation 4 , 5 . Furthermore, the ribosome is highly hydrated, due to the relatively open nature of RNA secondary and tertiary structure 6 .…”

Identification and Evaluation of Improved 4′- O -(Alkyl) 4,5-Disubstituted 2-Deoxystreptamines as Next-Generation Aminoglycoside Antibiotics

“…Aminoglycosides target bacterial protein synthesis by inducing codon misreading and by inhibiting translocation of the tRNA-mRNA complex ( 9 – 12 ). Aminoglycosides affect translation of the ribosome by direct interaction with decoding A-site rRNA.…”