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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 (metU(CUA)). 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 (metU(CUA/A1), metU(CUA/G72), and metU(CUA/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 metU(CUA/G72G73) tRNA to its glutaminylated form (by GatCAB) in M. smegmatis. In contrast, the glutamylated metU(CUA/G72) tRNA did not show a detectable level of transamidation. Interestingly, the metU(CUA/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.