Modified Nucleoside Dependent Watson−Crick and Wobble Codon Binding by tRNALysUUU Species

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Abstract
Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNALys3UUU with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASLLys3UUU), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNALys species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASLLys heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Ψ39), 5-methylaminomethyluridine (mnm5U34) and N6-threonylcarbamoyl-adenosine (t6A37). Incorporation of t6A37 and mnm5U34 contributed structure to the anticodon loop, apparent by increases in ΔS, and significantly enhanced the ability of ASLLys3UUU to bind poly-A programmed ribosomes. Neither ASLLys3UUU-t6A37 nor ASLLys3UUU-mnm5U34 bound AAG programmed ribosomes. Only the presence of both t6A37 and mnm5U34 enabled ASLLys3UUU to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNALys. The completely unmodified anticodon stem and loop of human tRNALys1,2CUU with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t6A37. The data suggest that tRNALysUUU species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.