[3P-53] Re-evaluating the biological function of an essential tRNA methyltransferase by experimental evolution
Post-transcriptional tRNA modifications improve the stability and decoding fidelity of tRNA and are essential for efficient translation. TrmD, which methylates the N1 atom of G37 in Pro, Arg, and Leu tRNAs, is one of several tRNA-modifying enzymes that are essential for growth in bacteria. Loss of TrmD increases the rate of +1 frameshift errors in translation of Pro codons, which is presumed to induce cell death. However, because bacteria are highly tolerant to translational frameshift errors, it is unclear why loss of TrmD is not tolerated. In this work, we performed experimental evolution of trmD knockdown strains of Escherichia coli to determine whether bacteria can evolve to tolerate loss of the m1G37 modification. trmD knockdown strains showed rapid growth recovery, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Our results suggest that inefficient aminoacylation of unmodified tRNA is responsible for lack of growth in the absence of the m1G37 modification and that ProS may function as a gatekeeper, preventing use of error-prone unmodified Pro-tRNA in translation. Our work shows the utility of experimental evolution for uncovering the biological functions of proteins, including essential enzymes.