Protein Synthesis Initiation in Bacteria
Formation of the ternary initiation complex of tRNAfMet-ribosome-mRNA requires that both the initiator tRNA and mRNA bind to their respective sites on the ribosome and that they form codon-anticodon hydrogen bonds with each other (, ). The ability of linezolid to disrupt initiation complex formation by inhibiting the binding of mRNA to the ribosome was examined. Figure shows that 200 μM linezolid did not inhibit the binding of a 200-bp synthetic mRNA to the ribosome.
11.4: Protein Synthesis (Translation) - Biology LibreTexts
Initiation factors IF1, IF2, and IF3 play important roles in the initiation of translation in bacteria. The tRNAfMet is bound by IF2 and is delivered to the 30S subunit joining IF1, IF3, and the mRNA as part of the initiation complex. Linezolid did not inhibit formation of the IF2-tRNAfMet complex when either 5 or 0.5 pmol of E. coli IF2 was used (Table ). The role of initiation factors in the mechanism of action of linezolid was further investigated by forming E. coli 70S ribosome initiation complexes in the absence of any of the three factors. Figure demonstrates that an IC50 of 152 μM was obtained for linezolid under these conditions.
Binding of 32P-labeled synthetic mRNA was carried out for 15 min at 24°C in duplicate 50-μl reaction mixtures containing 200 to 400 μg of 70S ribosomes, 20 mM MgCl2, 10 mM Tris-HCl (pH 7.4), 1 mM DTT, 80 mM NH4Cl, and 1 μl (16,000 dpm) of 32P-labeled mRNA. Duplicate reactions were terminated by the addition of 1 ml of ice-cold buffer containing 20 mM Tris (pH 7.4), 20 mM MgCl2, and 100 mM NH4Cl. The mRNA-ribosome complex was then trapped on Millipore HA filters, and the radioactivity was counted after the addition of scintillation fluid.
Antibiotics that Inhibit Bacterial Protein Synthesis
The oxazolidinones represent a new synthetic class of antibacterial agents with activity against gram-positive organisms (). Studies have shown that the oxazolidinones linezolid and eperezolid are active against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus faecium (, , , , ). Linezolid is entering phase III clinical testing as a therapeutic agent that is effective against skin and skin structure infections, bacteremia, and pneumonia caused by gram-positive pathogenic bacteria.
Initiation of protein synthesis: a target for antimicrobials ..
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 of N-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 from Escherichia coli. In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA to E. 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 the N-formylmethionyl-tRNA–ribosome–mRNA ternary complex.
Eukaryotic Protein Synthesis: Still a Mystery
After establishing that oxazolidinones inhibited initiation complex formation, further studies were designed to examine the effect of linezolid on isolated initiation events. IF2 forms a binary complex with tRNAfMet, guiding it to the P site of the ribosome where it can bond with the initiation codon of the mRNA (). High concentrations (200 μM) of linezolid did not inhibit binary complex formation, indicating that the binary complex reaction is probably not the target of oxazolidinones. After IF2 guides initiator tRNA to the P site, an initiation complex can be formed in the presence of mRNA. An mRNA sequence containing a strong Shine-Dalgarno site followed by seven bases, the AUG start codon, and a coding sequence was synthesized. To foster the formation of the initiation complex, the mRNA was constructed so as to minimize the potential secondary structure of the initiation site. With this [32P]mRNA with a defined sequence, it was demonstrated that 200 μM linezolid did not inhibit mRNA binding to ribosomes.