The Human Mitochondrial tRNAMet: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNAMetCAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNAMetCAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f5C34), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASLMetCAU), determined by NMR restrained molecular modeling, revealed how the f5C34 modification facilitates the decoding of AUA at the P- and the A-sites. The f5C34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASLMetCAU. The hmtASLMetCAU exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f5C34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f5C34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f5C34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.