Comparison of the misreading induced by streptomycin and neomycin

Grisé-Miron, Lucie; Noreau, Jean; Melançon, Pierre; Brakier‐Gingras, Léa

doi:10.1016/0005-2787(81)90032-0

Cited by 20 publications

(9 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Poly(U)-directed translation. The low-salt wash ribosomes and S100 preparation described above were used to study the effect of antibiotics on polyphenylalanine synthesis by the basic procedure of Grise-Miron et al (9). The reaction mixture contained in a total volume of 100 l 33 l of 3ϫ translation buffer (180 mM NH 4 Cl, 48 mM magnesium acetate, 189 mM Tris-HCl [pH 7.8], 48 mM ␤-mercaptoethanol, 3.9 mM ATP, 0.9 mM GTP, 51 mM phosphoenolpyruvate), 17 l of water, 1 l of 0.1 mM phenylalanine, 1 l of pyruvate kinase (5 mg/ml), 1 l of [2,3,4,5,6-3 H]phenylalanine, 10 l of phenylalanyl-tRNA (4 mg/ml in 5 mM potassium acetate buffer [pH 6.0]), 4 l of poly(U) RNA (4 mg/ml in 5 mM potassium acetate buffer [pH 6.0]), 5 l of unwashed ribosomes (1 A 260 U/l), and 5 l of 100,000 ϫ g supernatant (5 mg/ml).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions

Shinabarger¹,

Marotti²,

Murray³

et al. 1997

Antimicrob Agents Chemother

337

136

View full text Add to dashboard Cite

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

show abstract

Section: Methodsmentioning

confidence: 99%

Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions

Shinabarger¹,

Marotti²,

Murray³

et al. 1997

Antimicrob Agents Chemother

337

136

View full text Add to dashboard Cite

show abstract

“…5A). To assure the proper functionality of this assay neomycin, an aminoglycoside antibiotic known to increase the error rate during the decoding process, 38 was added to one reaction. Neither the single nucleobase deletion at 2421 nor the simultaneous base removal at 2421 and 2422 resulted in a higher rate of leucine misincorporation (Fig.…”

Section: Ribosomes Lacking the Guanine At 2421 Are Not Prone To Errorsmentioning

confidence: 99%

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis

Koch¹,

Clementi

Rusca³

et al. 2015

RNA Biology

View full text Add to dashboard Cite

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

show abstract

“…Streptomycin (25 pg/ml) was included for the culture of cells transformed with pPM523 (DHI-pPM523). Ribosomes were extracted from the bacterial pellets of these strains by high-speed centrifugation following standard procedures, and they were assayed for their misreading activity by monitoring the incorporation of isoleucine under the direction of poly(U) in the absence or the presence of streptomycin (detailed in 19). The Seguencing of the 16S rRNA region in the 530 looD.…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

A mutation in the 530 loop ofEscherichia coli16S ribosomal RNA causes resistance to streptomycin

Melançon¹,

Lemieux

Brakier‐Gingras³

1988

Nucl Acids Res

Self Cite

View full text Add to dashboard Cite

Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.

show abstract

Comparison of the misreading induced by streptomycin and neomycin

Cited by 20 publications

References 24 publications

Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions

Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis

A mutation in the 530 loop ofEscherichia coli16S ribosomal RNA causes resistance to streptomycin

Contact Info

Product

Resources

About