Impact of methylations of m²G966/m⁵C967 in 16S rRNA on bacterial fitness and translation initiation

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m ²G966 and m⁵C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m²G966/m⁵C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNA^fMet to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m²G966 and m ⁵C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.