Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNA(CUA/3GC) do not affect its formylation. However, the i-tRNA(CUA/3GC) is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmt(am274)), which affords initiation with i-tRNA(CUA/3GC). Replacement of fmt with fmt(am274) in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNA(CUA/3GC) into the fmt(am274) strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNA(CUA/3GC) causes a low level suppression of am274 in fmt(am274). Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNA(CUA/3GC) to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.