How does aminoglycosides inhibit protein synthesis

Toeprints were quantified and the extent of translocation determined, as described previously Shoji et al. Initial data fitting showed that the Hill coefficient fell between 0. Spontaneous reverse translocation was measured as detailed previously Shoji et al.

Briefly, mprogrammed ribosomes 0. Aliquots were removed at various time points and subjected to primer extension analysis. Fraction POST was quantified and plotted as a function of time. Data were fit to a single exponential function to obtain the observed rate and amplitude of the reaction. Ribosome recycling was measured using a double-membrane filtration method Fahlman and Uhlenbeck Ribosome-bound and free tRNA were captured by the top and bottom membranes, respectively Fahlman and Uhlenbeck ; Shoji et al.

The fraction of tRNA bound was quantified and plotted against time. Dissociation rate k off was determined by fitting the data to a single exponential function.

Then, k off was plotted as a function of AG concentration, and data were fit to a dose response equation analogous to the one described above, yielding the IC 50 value. Previous Section Next Section. Inhibition of translocation by aminoglycosides depends largely on the h44 site To determine the contribution of h44 and H69 binding sites to AG activities, we targeted each by mutagenesis.

View this table: In this window In a new window. TABLE 1. The ability of Neo to promote spontaneous reverse translocation is eliminated by h44 mutations It has been shown previously that Neo, Par, and Gen can similarly increase the rate of spontaneous reverse translocation Shoji et al.

Translocation assays EF-G-dependent translocation was measured by toeprinting Shoji et al. Ribosome recycling assay Ribosome recycling was measured using a double-membrane filtration method Fahlman and Uhlenbeck Previous Section. Deletion of a conserved, central ribosomal intersubunit RNA bridge. Mol Cell 23 : — Construction of Escherichia coli K in-frame, single-gene knockout mutants: the Keio collection.

Mol Syst Biol 2 : Nucleic Acids Res 42 : — CrossRef Medline Google Scholar. Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol 14 : — A steric block in translation caused by the antibiotic spectinomycin. ACS Chem Biol 2 : — Structural basis for hygromycin B inhibition of protein biosynthesis. RNA 14 : — Inhibition of ribosomal translocation by aminoglycoside antibiotics.

Biochem Biophys Res Commun 83 : — Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature : — The kinetic mechanism of bacterial ribosome recycling.

Nucleic Acids Res 45 : — CrossRef Google Scholar. Dallas A , Noller HF. Interaction of translation initiation factor 3 with the 30S ribosomal subunit.

Mol Cell 8 : — Misreading of RNA codewords induced by aminoglycoside antibiotics. Mol Pharmacol 1 : 93 — A further study of misreading of codons induced by streptomycin and neomycin using ribopolynucleotides containing two nucleotides in alternating sequence as templates. A role for the 30S subunit E site in maintenance of the translational reading frame.

RNA 15 : — Design and use of a fast and accurate in vitro translation system. In Ribosomes and protein synthesis—a practical approach ed. Spedding G , pp. Google Scholar. Contribution of the esterified amino acid to the binding of aminoacylated tRNAs to the ribosomal P- and A-sites. Biochemistry 43 : — Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.

Tobramycin variants with enhanced ribosome-targeting activity. Chembiochem 16 : — Fredrick K , Noller HF. Mol Cell 9 : — Catalysis of ribosomal translocation by sparsomycin.

Science : — Selection of the initiator tRNA by Escherichia coli initiation factors. Genes Dev 3 : — Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic.

EMBO J 21 : — Analysis of the contribution of individual substituents in 4,6-aminoglycoside-ribosome interaction. Antimicrob Agents Chemother 49 : — A genetic model to investigate drug-target interactions at the ribosomal decoding site. Biochimie 88 : — Binding of neomycin-class aminoglycoside antibiotics to mutant ribosomes with alterations in the A site of 16S rRNA. Antimicrob Agents Chemother 50 : — EMBO J 33 : — Ribosome recycling factor ribosome releasing factor is essential for bacterial growth.

Proc Natl Acad Sci 91 : — Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. EMBO J 17 : — Liu Q , Fredrick K. Contribution of intersubunit bridges to the energy barrier of ribosomal translocation. Nucleic Acids Res 41 : — Proc Natl Acad Sci : — Intersubunit bridges of the bacterial ribosome.

J Mol Biol : — Purification of active Escherichia coli ribosome recycling factor RRF from an osmo-regulated expression system. Biochimie 79 : — Magnet S , Blanchard JS. Molecular insights into aminoglycoside action and resistance.

Chem Rev : — Mol Cell 20 : — Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation. Biochem Biophys Res Commun 84 : — Correlated conformational events in EF-G and the ribosome regulate translocation. Nat Struct Mol Biol 17 : — Genetic analysis of new 16S rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus. J Antimicrob Chemother 66 : — Recognition of cognate transfer RNA by the 30S ribosomal subunit.

Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell : — Insights into the decoding mechanism from recent ribosome structures. Trends Biochem Sci 28 : — Kinetically competent intermediates in the translocation step of protein synthesis. Mol Cell 25 : — Nat Commun 5 : Sequence of steps in ribosome recycling as defined by kinetic analysis. Mol Cell 18 : — Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation.

RNA 13 : — Aminoglycoside resistance with homogeneous and heterogeneous populations of antibiotic-resistant ribosomes. Antimicrob Agents Chemother 45 : — Structure-activity relationships among the kanamycin aminoglycosides: role of ring I hydroxyl and amino groups.

Antimicrob Agents Chemother 56 : — Structure-based energetics of mRNA decoding on the ribosome. Biochemistry 53 : — Molecular insights into aminoglycoside action and resistance.

Sutcliffe, J. Improving on nature: antibiotics that target the ribosome. Davies, J. Misreading of RNA codewords induced by aminoglycoside antibiotics. CAS Google Scholar. A further study of misreading of codons induced by streptomycin and neomycin using ribopolynucleotides containing two nucleotides in alternating sequence as templates.

Inhibition of ribosomal translocation by aminoglycoside antibiotics. Article Google Scholar. Misumi, M. Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation. Hirokawa, G. Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic. EMBO J. Wilson, D. The how and Y of cold shock.

Janosi, L. Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. Zavialov, A.

Cell 18 , — Gao, N. Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies. Peske, F. Sequence of steps in ribosome recycling as defined by kinetic analysis. The ribosome-recycling step: consensus or controversy? Trends Biochem. The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits.

RNA 11 , — Teyssier, E. Temperature-sensitive mutation in yeast mitochondrial ribosome-recycling factor RRF. Nucleic Acids Res. Rolland, N. Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in Escherichia coli carrying temperature-sensitive ribosome recycling factor.

USA 96 , — Ribosome recycling factor ribosome releasing factor is essential for bacterial growth. USA 91 , — Campuzano, S. Functional interaction of neomycin B and related antibiotics with 30S and 50S ribosomal subunits. Carter, A. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature , — Selmer, M. Science , — Schuwirth, B. Structures of the bacterial ribosome at 3.

Moazed, D. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Yusupov, M. Crystal structure of the ribosome at 5. Cannone, J. BMC Bioinformatics 3 , 2 Article PubMed Google Scholar.

Lancaster, L. Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing. Cell , — Valle, M.

Locking and unlocking of ribosomal motions. Agrawal, R. Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications. USA , — Connell, S. Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors. Cell 25 , — Raj, V. X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit.

Ali, I. Deletion of a conserved, central ribosomal intersubunit RNA bridge. Cell 23 , — Liiv, A. Analysis of the function of E. BMC Mol. Rackham, O. Functional epitopes at the ribosome subunit interface. Frank, J. A ratchet-like intersubunit reorganization of the ribosome during translocation. Horan, L. Intersubunit movement is required for ribosomal translocation. Dissociation of guanosine nucleotide-elongation factor G-ribosome complexes.

Biochemistry 18 , — Senior, K. Duchenne muscular dystrophy improved by gentamicin. Today 5 , Otwinowski, Z. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. Collaborative Computational Project, Number 4.

The CCP4 suite: programs for protein crystallography.