[46] Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli

Ettayebi, Mohamed; Borden, Angela; Morgan, Edward A.

doi:10.1016/s0076-6879(88)64077-8

Cited by 187 publications

(168 citation statements)

References 7 publications

Supporting

Mentioning

164

Contrasting

Unclassified

Order By: Relevance

“…The ability of pLK35 or its mutant derivatives to displace prrnS12 was tested by growing MC250 transformants on medium containing streptomycin, which permits growth only of those cells that have lost prrnS12 and express rRNA exclusively from the pLK35 plasmid. Exclusive expression of mutant rRNA in the resulting strains was confirmed by primer extension analysis of total RNA (19).…”

Section: Methodsmentioning

confidence: 93%

“…DH1 pCI857 pGQ7 (wild-type) and derivatives encoding the three 2451 mutations were grown to saturation at 30°, diluted into fresh medium and grown at 42°C for 2.5 h. Cell lysates were fractionated through sucrose gradients as described (20,21). Gradient fractions containing 50S subunits, 70S ribosomes, or polysomes were collected, and rRNA was extracted and analyzed by primer extension with a primer complementary to residues 2450-2469 of 23S rRNA (19). Relative intensity of bands corresponding to extension products from the mutant and wild-type templates was determined with a PhosphorImager (Molecular Dynamics) and IMAGEQUANT software.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

Thompson

Kim

O’Connor

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

184

116

View full text Add to dashboard Cite

On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length ␤-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3-to 14-fold by these mutations. Peptidyltransferase activity and in vitro ␤-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.

show abstract

Section: Methodsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

Thompson

Kim

O’Connor

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

184

116

View full text Add to dashboard Cite

show abstract

“…The ratio of mutant to WT 16S rRNA in the gradient fractions was determined by primer extension as described in ref. 3, using a primer, AAGGGCCATGATGACTTGA, specific for the pLK45 resident mutation C1192U. Primer-extension products were separated on 12% denaturing polyacrylamide gel and quantified by using a PhosphorImager (Molecular Dynamics).…”

Section: Methodsmentioning

confidence: 99%

“…Advantages of the ribosome as an antibiotic target may stem from its RNA-based design. The multiplicity of rRNA genes in microbial genomes makes it difficult for a microorganism to develop resistance by mutating the drug-binding site (3). Furthermore, RNA offers fewer mutational options than protein enzymes (3 versus 19, respectively), which makes it more difficult for a microbial pathogen to ''find'' a mutation that would reduce antibiotic binding without compromising functional integrity of the enzyme.…”

mentioning

confidence: 99%

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Yassin

Fredrick

Mankin

2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Many clinically useful antibiotics interfere with protein synthesis in bacterial pathogens by inhibiting ribosome function. The sites of action of known drugs are limited in number, are composed primarily of ribosomal RNA (rRNA), and coincide with functionally critical centers of the ribosome. Nucleotide alterations within such sites are often deleterious. To identify functional sites and potential sites of antibiotic action in the ribosome, we prepared a random mutant library of rRNA genes and selected dominant mutations in 16S rRNA that interfere with cell growth. Fifty-three 16S rRNA positions were identified whose mutation inhibits protein synthesis. Mutations were ranked according to the severity of the phenotype, and the detrimental effect of several mutations on translation was verified in a specialized ribosome system. Analysis of the polysome profiles of mutants suggests that the majority of the mutations directly interfered with ribosome function, whereas a smaller fraction of mutations affected assembly of the small ribosomal subunit. Twelve of the identified mutations mapped to sites targeted by known antibiotics, confirming that deleterious mutations can be used to identify antibiotic targets. About half of the mutations coincided with known functional sites in the ribosome, whereas the rest of the mutations affected ribosomal sites with less clear functional significance. Four clusters of deleterious mutations in otherwise unremarkable ribosomal sites were identified, suggesting their functional importance and potential as antibiotic targets.16S rRNA ͉ 30S subunit ͉ resistance ͉ ribosome ͉ translation W ith a molecular mass of Ϸ2.5 million Da and Ͼ50 different RNA and protein building blocks, the ribosome represents one of the largest and most complex enzymes in the cell. Ribosomal RNA (rRNA) accounts for two-thirds of the ribosome and is responsible for its main functions in protein synthesis: interpretation of genetic information and polymerization of amino acids into a polypeptide. rRNA is also intimately involved in known auxiliary activities of the ribosome, such as nascent peptide release, binding of translation factors, GTP hydrolysis, etc. (reviewed in ref. 1).The ribosome is the predominant antibiotic target in the bacterial cell. A large variety of natural and synthetic antibiotics interfere with translation by binding to rRNA and preventing the correct placement of ribosomal ligands, corrupting rRNA structure, or affecting conformational flexibility of rRNA (2). Advantages of the ribosome as an antibiotic target may stem from its RNA-based design. The multiplicity of rRNA genes in microbial genomes makes it difficult for a microorganism to develop resistance by mutating the drug-binding site (3). Furthermore, RNA offers fewer mutational options than protein enzymes (3 versus 19, respectively), which makes it more difficult for a microbial pathogen to ''find'' a mutation that would reduce antibiotic binding without compromising functional integrity of the enzyme. In the clinical setting, ...

show abstract

“…Cultures were grown in LB medium (5 g NaCl/L), 200 mg mL ampicillin, at either 30 8C or 42 8C to an A 430 of 0+8 (N.B+: for cultures of PEM100 pSTL102 and PEM100 pKK1400U/2058G grown at 42 8C a large inoculum was used to reach an A 430 of 0+8 within approximately 3 h)+ Cells were collected by centrifugation and ribosomes and total cellular RNA extracted (Godson & Sinsheimer, 1967)+ Polysomes, 70S, 50S, and 30S ribosomal fractions were separated by sucrose density gradient centrifugation (De Stasio & Dahlberg, 1990), and the mutant and wild-type rRNAs were extracted and analyzed by primer extension (Sigmund et al+, 1988)+…”

Section: Ribosomal Rna Analysismentioning

confidence: 99%

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

Koosha

Cameron

Andrews

et al. 2000

RNA

View full text Add to dashboard Cite

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.

show abstract

[46] Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli

Cited by 187 publications

References 7 publications

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

Contact Info

Product

Resources

About