Polysomes Extracted from
            <i>Escherichia coli</i>
            by Freeze-Thaw-Lysozyme Lysis

Ron, Eliora Z.; Kohler, Robert Ε.; Davis, Bernard D.

doi:10.1126/science.153.3740.1119

Cited by 177 publications

(96 citation statements)

References 5 publications

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“…Polysomes were analyzed following published protocols (20), with minor modifications described in Supporting Materials and Methods. The ratio of mutant to WT 16S rRNA in the gradient fractions was determined by primer extension as described in ref.…”

Section: Methodsmentioning

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.

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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, ...

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Section: Methodsmentioning

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.

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“…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).…”

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.

187

116

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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.

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“…Polysome Profile Analysis-Polysomes were analyzed as described previously (32,33) with minor modifications as described in Ref. 25.…”

Section: Mutagenesis and Construction Of Segment-mutant Libraries-mentioning

confidence: 99%

Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

Yassin¹,

Mankin

2007

Journal of Biological Chemistry

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The ribosome is the main target for antibiotics that inhibit protein biosynthesis. Despite the chemical diversity of the known antibiotics that affect functions of the large ribosomal subunit, these drugs act on only a few sites corresponding to some of the known functional centers. We have used a genetic approach for identifying structurally and functionally critical sites in the ribosome that can be used as new antibiotic targets. By using randomly mutagenized rRNA genes, we mapped rRNA sites where nucleotide alterations impair the ribosome function or assembly and lead to a deleterious phenotype. A total of 77 single-point deleterious mutations were mapped in 23 S rRNA and ranked according to the severity of their deleterious phenotypes. Many of the mutations mapped to familiar functional sites that are targeted by known antibiotics. However, a number of mutations were located in previously unexplored regions. The distribution of the mutations in the spatial structure of the ribosome showed a strong bias, with the strongly deleterious mutations being mainly localized at the interface of the large subunit and the mild ones on the solvent side. Five sites where deleterious mutations tend to cluster within discrete rRNA elements were identified as potential new antibiotic targets. One of the sites, the conserved segment of helix 38, was studied in more detail. Although the ability of the mutant 50 S subunits to associate with 30 S subunits was impaired, the lethal effect of mutations in this rRNA element was unrelated to its function as an intersubunit bridge. Instead, mutations in this region had a profound deleterious effect on the ribosome assembly.

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Polysomes Extracted from Escherichia coli by Freeze-Thaw-Lysozyme Lysis

Abstract: Polysomes can be extracted from Escherichia coli by freezing and thawing in the presence of lysozyme, followed by treatment with sodium deoxycholate. The method is simple and convenient; the yields consistently high.

Cited by 177 publications

References 5 publications

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

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

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

Potential New Antibiotic Sites in the Ribosome Revealed by Deleterious Mutations in RNA of the Large Ribosomal Subunit

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