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

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein 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.