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 assay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporter systems to study translation initiation and elongation in The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA, ) molecules containing a CUA anticodon (). 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 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 (, , and ) with a Watson-Crick base pair at the 1·72 position participate in elongation.

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.