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 tRNAsLeu(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 tRNAsLeu(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 tRNAArg 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.