The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging ofN-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits fromEscherichia coli. In addition, complex formation withStaphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA toE. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2–N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of theN-formylmethionyl-tRNA–ribosome–mRNA ternary complex.

A soluble protein factor was isolated, free of elongation factor (EF-T and EF-G, based on its ability to stimulate the synthesis of eptide bonds using ribosomal bound 7OS-AUG.N-formyl-34S methionyl-tRNA complex and added puromycin as substrates. Over 90% of this activity was found in the ribosome-free cytoplasm of Escherichia coli extracts. Other features such as molecular weight, purification properties, and catalytic activities distinguish this factor from ribosomal proteins and known activators of translation. The factor requires all components needed for peptide bond synthesis and is inhibited by antibiotics known to specifically block the peptidyl transferase activity of ribosomes. The factor increases the binding affinity of the ribosome for the aminoacyl-tRNA analog puromycin about 10-fold. We suggest that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and regard it as a new factor required for peptide chain elongation, which we call EF-P.A functional topology of ribosomes has been constructed to accommodate our intuition of how these particles might catalyze peptide bond synthesis. Peptide chain synthesis, as presently conceived, is a cyclical process that takes place on the ribosome. It involves a site specific alignment between peptidyl-tRNA and aminoacyl-tRNA, peptide bond formation, and translocation of mRNA. The binding of aminoacyltRNA and the translocation of mRNA are mediated by the soluble proteins elongation factor (EF)-T (Tu + Ts) and EF-G, respectively, and require GTP hydrolysis (1). Until now the formation of peptide bonds has been thought to proceed "spontaneously" without the requirement for soluble factors or exogenous energy sources (1, 2). However, we now report that the reaction between ribosomal bound Nformyl-Met-tRNA and puromycin, the classical model of peptide bond synthesis, is indeed stimulated by a soluble protein factor, which we have called EF-P. Thus peptide bond synthesis need not be restricted to an autonomous function of the ribosome.The relationship of this protein, EF-P, to component X, a factor needed for translation with messengers other than poly(U) (3-5), is discussed. RESULTS AND DISCUSSIONPeptide bond synthesis between puromycin, an analog of the amino acid terminus of aminoacyl-tRNA, and ribosomal bound N-formyl-Met-tRNA is catalyzed by 70S or 50S particles, and is reported to occur without soluble factors or GTP (2, 6). However, as shown in Table 1, when the puromycin concentration is lowered to 1-3 AM, adding the soluble factor results in a marked stimulation of N-formyl-[%5S]Metpuromycin synthesis. To simplify discussion we refer to this factor as EF-P, i.e., a factor that stimulates the peptidyl transferase reaction.Abbreviation: EF, elongation factor. EF-P activity may have gone undetected thus far (6) because at the levels of puromycin generally used to assay peptidyl transferase activity the ribosomes are saturated with this analog, and the "spontaneous' formation of N-formylmethionyl-p...

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A.Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (Kan R ) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA ؉ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-Kan R gene in the chromosome grew at 33°C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44°C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44°C. At 44°C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.The most important catalytic function of the ribosome is the synthesis of peptide bonds. A variety of approaches have been used to deduce the components that comprise this catalytic center. The results of in vitro reconstitution studies, photochemical cross-linking of substrates, and mutagenesis of conditionally lethal or antibiotic-resistant phenotypes have implicated domain V of the 23 S rRNA as well as proteins L2, L3, and L4 as the minimum components of this active center (1-6).A surprising finding is that the in vitro reconstituted peptidyltransferase cannot condense all aminoacyl-tRNA template combinations (7). This anomaly is reflected in the fact there is a subsite on domain V of 23 S rRNA that is specific for hydrophobic amino acids (2). In retrospect, it has been known for more than two decades that puromycin, which is one of the most common substrate analogues used to study this reaction, has a special three-dimensional structure (a U shape) that favors peptide-bond synthesis (8). Substitution of the aromatic residue of puromycin by that of other amino acids distorts this structure and drastically impairs peptide-bond synthesis (9). This specificity is reflected in the 50 S catalyzed "fragment" reaction that has been used to deduce the components of the peptidyltransferase catalytic center.Reconstitution studies as well as photoaffinity labeling experiments indicate that several proteins of the 50 S particle enhance peptide-bond synthesis. The assembled peptidyltransferase in the 70 S ribosome catalyzes peptide bonds at a higher rate than does the peptidyltransferase of the 50 S subunit, but does not efficiently condense nonaromatic amino acids (7, 10). In addressing this issue, we asked whether proteins that stimulate reconstitution of translation from homogeneous translation factors enhance the condensation of ...

Elongation factor EF‐P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50‐S ribosomal subunit. This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine‐puromycin. In the present work, we tested the ability of EF‐P to promote peptide bond synthesis between ribosome‐bound fMet‐tRNA and several analogues of the 3′ terminus of aminoacyl‐tRNA, i.e. the cytidylyl(3′‐5′)‐[2′(3′)‐O‐l‐aminoacyladenosines]. EF‐P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction. This activity of EF‐P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF‐Tu, EF‐Ts and EF‐G. One role of EF‐P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl‐tRNAs that are poor acceptors for the ribosomal peptidyl transferase.

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMettRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.A novel class of antimicrobial agents, the oxazolidinones, target a wide spectrum of gram-positive and anaerobic bacteria (4, 6, 9, 28). These compounds act by inhibiting protein synthesis and have no effect on replication or transcription (8). Cell extracts exposed to oxazolidinones are impaired in protein synthesis when programmed by native mRNAs but do not appear to be inhibited when programmed by synthetic mRNAs that lack the signals required for initiation and termination of translation (7,8,26). This suggested that these compounds may target the initiation reaction. The oxazolidinone PNU-100766 (linezolid; Fig. 1) inhibits binding of the initiator fMettRNA to the 70S ribosomal particle programmed with a synthetic mRNA that harbors a Shine-Dalgarno sequence and a properly spaced initiation codon (29).Mutations to oxazolidinone resistance map to domain V of the 23S rRNA in Halobacterium halobium (18), Staphylococcus aureus (27), and the enterococci while mapping to domains IV and V in Escherichia coli (33). The position of these PNU-100766 resistance mutations suggested to us that the oxazolidinones may target peptidyl transferase indirectly by affecting the binding of the initiator tRNA. Since the P site accommodates the initiator tRNA and the nascent protein, these drugs could also affect the affinity of the peptidyl-tRNA for the ribosome.Recent studies have indicated that the oxazolidinones bind to 70S ribosomes, as well as to 50S subunits (19), but not to 30S subunits. In contrast, a report by Matassova et al. (20) demonstrated that oxazolidinone footprints map to the central domain of the 16S rRNA whereas the 23S rRNA ...