Evolutionary repair reveals an unexpected role of the tRNA modification m¹G37 in aminoacylation

The tRNA modification m(1)G37, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it suppresses translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m(1)G37 is not tolerated. Here, we addressed this question through experimental evolution of trmD mutant strains of Escherichia coli. Surprisingly, trmD mutant strains were viable even if the m(1)G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via duplication or mutation of the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNA(Pro) is aminoacylated less efficiently than m(1)G37-modified tRNA(Pro), and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNA(Pro). These results show that inefficient aminoacylation of tRNA(Pro) is the main reason for growth defects observed in trmD mutant strains and that proS may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNA(Pro) in translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.