Modified uridines with C5-methylene substituents at the first position of the tRNA anticodon stabilize U·G wobble pairing during decoding

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm⁵U) at the wobble position. However, the functional properties of the C5-substituents of xm⁵U in codon recognition remain elusive. We previously found that mitochondrial tRNAs ^Leu(UUR) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (τm⁵U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs^Leu(UUR) with or without xm⁵U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA^Arg lacking mnm⁵U modification in a knock-out strain of the modifying enzyme (ΔmnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm⁵U modification plays a critical role in decoding NNG codons by stabilizing U·G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing τm⁵U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the τm⁵U·G base pair does not have classical U·G wobble geometry. These structures provide help to explain how the τm⁵U modification enables efficient decoding of UUG codons.