Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading

Abstract

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.Streptomycin, an error-enhancing aminoglycoside antibiotic, perturbs translational fidelity primarily by interfering with the proofreading control (39,42,48). The proofreading control of translational accuracy triggers the rejection of near-cognate tRNAs which escaped the initial discrimination step of tRNA decoding. According to the two-step model for tRNA decoding, the initial discrimination step is mediated by the reversible binding of the ternary complex of aminoacyl-tRNA.EF-Tu.GTP to the ribosome, and the proofreading control takes place following hydrolysis of the GTP complexed to elongation factor Tu (EF-Tu). Thompson and coworkers have shown that proofreading efficiency depends upon the rate of dissociation of EF-Tu.GDP from the ribosome (reviewed in reference 47); the lower this rate, the higher the probability of rejecting a near-cognate tRNA before allowing the incorporation of the aminoacyl residue into the peptide bond. This view has been challenged by Ehrenberg et al. (13), who think that EF-Tu.GDP has left the ribosome before proofreading of tRNA. Although EF-Tu interacts with the 30S subunit, the 50S subunit provides the major determinants for EF-Tu binding (reviewed in references 28 and 34). One can thus assume that the 50S subunit should play an important part in proofreading control.Attempts to understand the interference of streptomycin with proofreading control have focused on 30S mutants which fail to bind the drug and consequently abolish its misreading effect. This was the case for the classical streptomycin-resistant mutants with altered ribosomal protein S12 (35) and for mutations in the 915 region or the stem-loop 530 region of 16S rRNA (25,26,38). We have hypothesized that it should be possible to obtain mutants which, while still binding the drug, would decrease its effect. In this study, we have investigated whether mutations in the 2660 loop of 23S rRNA interfere with the action of streptomycin. This loop is known to be located in the binding site of 27,33), which makes it a likely candidat...

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Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus

“…It is notable that translational accuracy can also be affected by two components of the 50S subunit, ribosomal protein L6 and the 2660 loop region of the 23S rRNA. Mutations have been identified in these components that result in a decreased translation rate, greater accuracy of protein synthesis, and increased resistance to many of the misreading-inducing aminoglycoside antibiotics, in particular, gentamicin (17,26).…”

Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

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