Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria.

Genetic analysis of the mechanism of protein synthesis in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based in vivo reporter systems to study translation initiation and elongation in Mycobacterium smegmatis The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA, metU) molecules containing a CUA anticodon (metUCUA). The assay systems allow structure-function analyses of tRNAs without interfering with the cellular protein synthesis and function with or without the expression of heterologous GlnRS from Escherichia coli We show that despite their naturally occurring slow-growth phenotypes, the step of i-tRNA formylation is vital in translation initiation in mycobacteria and that formylation-deficient i-tRNA mutants (metUCUA/A1, metUCUA/G72, and metUCUA/G72G73) with a Watson-Crick base pair at the 1·72 position participate in elongation. In the absence of heterologous GlnRS expression, the mutant tRNAs are predominantly aminoacylated (glutamylated) by nondiscriminating GluRS. Acid urea gels show complete transamidation of the glutamylated metUCUA/G72G73 tRNA to its glutaminylated form (by GatCAB) in M. smegmatis In contrast, the glutamylated metUCUA/G72 tRNA did not show a detectable level of transamidation. Interestingly, the metUCUA/A1 mutant showed an intermediate activity of transamidation and accumulated in both glutamylated and glutaminylated forms. These observations suggest important roles for the discriminator base position and/or a weak Watson-Crick base pair at 1·72 for in vivo recognition of the glutamylated tRNAs by M. smegmatis GatCAB.IMPORTANCE Genetic analysis of the translational apparatus in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporters which utilize specific decoding of amber codons by mutant tRNAs at the steps of initiation and/or elongation to allow structure-function analysis of the translational machinery. We show that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-tRNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS. The discriminator base position, and/or a weak Watson-Crick base pair at the top of the acceptor stem, provides important determinants for transamidation of the i-tRNA-attached Glu to Gln by the mycobacterial GatCAB.

Antimicrobial activity of fusidic acid in Escherichia coli is dependent on the relative levels of ribosome recycling factor and elongation factor G.

During protein synthesis, elongation factor G (EFG) participates at the steps of translocation and ribosome recycling. Fusidic acid (FA) is a bacteriostatic antibiotic, which traps EFG on ribosomes, stalling them on mRNAs. How the bacterial susceptibility to FA is determined, and which of the two functions of EFG (translocation or ribosome recycling) is more vulnerable, has remained debatable. The in vivo studies addressing these aspects of FA mediated inhibition of protein synthesis are lacking. Here, we used a system of Escherichia coli strains and their complementation/supplementation with the plasmid borne copies of the inducible versions of EFG and ribosome recycling factor (RRF) genes. Additionally, we investigated FA sensitivity in a strain with increased proportion of stalled ribosomes. We show that the cells with high EFG/RRF (or low RRF/EFG) ratios are more susceptible to FA than those with low EFG/RRF (or high RRF/EFG) ratios. Our in vivo observations are consistent with the recent in vitro reports of dependence of FA susceptibility on EFG/RRF ratios, and the notion that an overriding target of FA is the translocation function of EFG. An applied outcome of our in vivo study is that FA mediated growth inhibition could be facilitated by depletion or inactivation of cellular RRF.