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

The functional centers of the ribosome in all organ-isms 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 essen-tial for growth and hence the functional role of modi-fications is largely unknown. Here, we show that the methyl groups of nucleosides m2G966 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 modi-fications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNAfMet to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m2G966 and m5C967 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