Modified Nucleoside Dependent Watson-Crick and Wobble Codon Binding by tRNA super(Lys) sub(UUU) Species

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA super(Lys3) sub(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL super(Lys3) sub(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA super(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL super(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine ( psi sub(39)), 5-methylaminomethyluridine (mnm super(5)U sub(34)) and N6-threonylcarbamoyl-adenosine (t super(6)A sub(37)). Incorporation of t super(6)A sub(37) and mnm super(5)U sub(34) contributed structure to the anticodon loop, apparent by increases in Delta S, and significantly enhanced the ability of ASL super(Lys3) sub(UUU) to bind poly-A programmed ribosomes. Neither ASL super(Lys3) sub(UUU)-t super(6)A sub(37) nor ASL super(Lys3) sub(UUU)-mnm super(5)U sub(34) bound AAG programmed ribosomes. Only the presence of both t super(6)A sub(37) and mnm super(5)U sub(34) enabled ASL super(Lys3) sub(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA super(Lys). The completely unmodified anticodon stem and loop of human tRNA super(Lys1,2) sub(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t super(6)A sub(37). The data suggest that tRNA super(Lys) sub(UUU) 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.