A mutation in the 530 loop of<i>Escherichia coli</i>16S ribosomal RNA causes resistance to streptomycin

Melançon, Pierre; Lemieux, Claude; Brakier‐Gingras, Léa

doi:10.1093/nar/16.20.9631

Cited by 93 publications

(54 citation statements)

References 38 publications

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“…The low transformation linkage with rpsE suggests that the location of strR is downstream of a, which is contiguous with spc, and probably downstream of the genes for S9 and S20, which show higher linkage to rpsE. The strR mutation could affect genes known to be located in (11,20); in general, the resistance phenotype is observed only when the mutant rrn operon is overexpressed, since there are 7 copies of rrn operons in E. coli (9) and 10 in B. subtilis (19,30). If the strR mutation is an alteration in one of the rrn operons located in this region, then this operon must be expressed at a much higher level than other rrn operons.…”

Section: Discussionmentioning

confidence: 99%

Bacillus subtilis mutants with alterations in ribosomal protein S4

1990

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Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsDI and rpsD2, were located between the argGH and aroG genes, at 2630 on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 120. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsDI allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.A powerful strategy in characterization of the structure and function of the bacterial ribosome is the isolation and analysis of mutants with alterations in ribosomal components. In previous studies, we reported the isolation and characterization of a streptomycin-resistant (Strr)J, sporeminus (Spo-) mutant of Bacillus subtilis (4, 14) and secondsite revertants in which the Spo-phenotype was suppressed. Several of these revertants exhibited changes in the electrophoretic mobility of individual ribosomal proteins (13). The original Strr Spo-strain contains two mutations, one of which, designated rpsL2, affects the 30S subunit ribosomal protein S12; the second, designated strR, affects an unidentified 30S subunit component (14). One revertant strain contained an additional mutation that causes an alteration in the electrophoretic mobility of ribosomal protein S4. This mutation, designated rpsDl, maps to position 2630 on the B. subtilis chromosome, between argGH (2600) and aroG (264°; 23), distant from the major cluster of ribosomal protein genes at 120 (15). Although the rpsDl mutation was presumed to be a lesion in the structural gene encoding ribosomal protein S4, it was also possible that the mutation affected a gene involved in the posttranslational modification of S4.In this study, a second mutant with a different alteration in the electrophoretic mobility of protein S4 was isolated by using a similar selection procedure. Since this mutation presumably causes a different structural alteration in protein S4, analysis of the mutations at the primary structural level would provide strong evidence as to whether they affect the S4 structural gene. We report (i) the isolation and characterization of the second S4 mutant and (ii) the cloning and analysis of both mutant alleles. The results presented show that both mutations do in fact represent alterations in rpsD, the gene encoding S4, and provide information about the role of S4 in the...

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Section: Discussionmentioning

confidence: 99%

Bacillus subtilis mutants with alterations in ribosomal protein S4

1990

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“…The mutations were introduced into the EcoRIBamHI fragment of the rrnB operon cloned in phage M13, as described previously (45), by the method of Kunkel (22). Plasmid pPM523, a derivative of pKK3535 with an A-*C substitution at position 523 of 16S rRNA (29), was used for comparison. The lacZ plasmids used in the nonsense suppression and frameshifting experiments were a generous gift from Michael O'Connor, Brown University, Providence, R.I.…”

Section: Methodsmentioning

confidence: 99%

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

1992

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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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“…Despite this difference, streptomycin binds at the functional center of the ribosome in close proximity to the binding site of paromomycin. Tight binding of antibiotic is facilitated by interaction with nucleotides 13, 526, 915, and 1490 of 16S rRNA and protein S12 (45, 105,186,191,196). Like 2-deoxystreptamine-containing aminoglycosides, streptomycin induces misreading of the genetic code (142,143,250), but the underlying mechanism seems to be different.…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Versatility of Aminoglycosides and Prospects for Their Future

2003

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Aminoglycoside antibiotics have had a major impact on our ability to treat bacterial infections for the past half century. Whereas the interest in these versatile antibiotics continues to be high, their clinical utility has been compromised by widespread instances of resistance. The multitude of mechanisms of resistance is disconcerting but also illuminates how nature can manifest resistance when bacteria are confronted by antibiotics. This article reviews the most recent knowledge about the mechanisms of aminoglycoside action and the mechanisms of resistance to these antibiotics

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

Cited by 93 publications

References 38 publications

Bacillus subtilis mutants with alterations in ribosomal protein S4

Bacillus subtilis mutants with alterations in ribosomal protein S4

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

Versatility of Aminoglycosides and Prospects for Their Future

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