No abstract

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.During the course of evolution, a disproportionately large number of natural antibiotics have been selected to act upon the ribosome. In the majority of cases, these drugs bind to ribosomes by interacting directly with rRNA (8). Due to the presence of multiple copies of rRNA genes in most species, it is difficult for a sensitive organism to develop resistance by mutating the antibiotic binding site, which is probably one of the main reasons why the ribosome has been repeatedly selected as an antibiotic target.Conditions created by the extensive and sometimes uncontrolled use of natural and synthetic antibiotics for antimicrobial therapy have promoted the selection and rapid spread of resistant pathogens that exhibit high tolerance to many drugs, including those which are targeted against the ribosome. Although the occurrence of antibiotic resistance mutations in rRNA genes is fairly rare in comparison with other types of resistance, a number of such cases have been reported, especially in those pathogens which contain only one or two copies of rRNA operons in their chromosome (B. Vester and S. Douthwaite, submitted for publication).The rapidly growing incidence of drug resistance in pathogenic bacteria urges the development of new antibiotics. Several new drugs targeted against the ribosome are currently being developed, including the oxazolidinones (3, 18). After first being identified as prospective antimicrobial agents in 1987 (32), oxazolidinones were abandoned for some time due to their high toxicity. Later on, new derivatives with superior pharmacological properties were found (3, 16), and recently one of the oxazolidinone antibiotics, linezolid (Fig. 1A), has ...

It was assumed for a long time that the ribosome is indifferent to the sequence of the polypeptide it is synthesizing. New evidence, however, indicates that nascent or newly synthesized polypeptides can affect functions of the ribosome in cis. In a number of cases, the newly translated peptide exerts its effect on translation while still being located within the ribosome. For example, short nascent peptides regulate stalling of the ribosome on mRNA, which is required for inducing the expression of chloramphenicol resistance (cat and cmlA) and erythromycin resistance (erm) genes (1, 2). Ribosome stalling depends on the amino acid sequence of the nascent peptide rather than on the nucleotide sequence of mRNA and occurs when the nascent peptide is only several amino acids long and should be located within the ribosome. Other examples include: translational bypass of the coding gap in bacteriophage T4 gene 60 mRNA (ribosome "hopping"), which depends on the amino acid sequence of the nascent peptide (3); dependence of termination efficiency on the amino acid sequence of the nascent peptide (4); attenuation of eukaryotic gene expression by short upstream open reading frames, which depends on the encoded amino acid sequences but not the mRNA sequence; and others (5-7). Despite a growing number of cases where cis-action of the newly synthesized peptide on the ribosome has been either demonstrated or suspected, almost nothing is known about molecular mechanisms of interaction between the ribosome and regulatory cis-acting peptides. The nature and location of the peptideresponsive site remains obscure.A new example of a cis-acting peptide is represented by a pentapeptide encoded in Escherichia coli 23 S rRNA (6). It was demonstrated that production in E. coli cells of a 34-nucleotidelong segment of 23 S rRNA, positions 1235-1268 (8), renders cells resistant to the ribosome-targeted antibiotic erythromycin (8, 9). Curiously, erythromycin resistance was mediated by translation of a pentapeptide (E-peptide) 1 encoded in the rRNA fragment. Mutations that affected translation initiation signals of the E-peptide mini-gene (Shine-Dalgarno region and initiator GUG codon) abolished erythromycin resistance. Interestingly, mutations at the terminator UAA codon, as well as some missense mutations, also interfered with peptide activity, suggesting that the size of the peptide and its amino acid sequence are essential for its functions. Translation of the E-peptide mRNA in the cell-free system rendered ribosomes resistant to erythromycin. However, addition of the synthetic E-peptide to the translating ribosome in vitro did not confer any erythromycin resistance (8). Thus, it appears that the E-peptide acts in cis so that only the ribosome on which the peptide has been translated becomes resistant to the drug. The single binding site of erythromycin is located on the large ribosomal subunit in the vicinity of the peptidyltransferase center. Accordingly, one possible mechanism of the E-peptide action is that the newly translated peptide ...

Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA. Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits). Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA. rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.