Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNA(UUU)(Lys) species

Nucleoside modifications are important to the structure of all tRNAs and ar e critical to the function of some tRNA species. The transcript of human tR NA(UUU)(Lys3) With a UUU anticodon, and the corresponding anticodon stem an d loop domain (ASL(UUU)(Lys3)), are unable to bind to poly-A programmed rib osomes. To determine if specific anticodon domain modified nucleosides of t RNA(Lys) species would restore ribosomal binding and also affect thermal st ability, we chemically synthesized ASL(Lys) heptadecamers and site-specific ally incorporated the anticodon domain modified nucleosides pseudouridine ( Psi (39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoy l-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contrib uted structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(UUU)(Lys3) to bind poly-A program med ribosomes. Neither ASL(UUU)(Lys3)-t(6)A(37) nor ASL(UUU)(Lys3)-mnm(5)U( 34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(UUU)(Lys3) to bind AAG programmed ribosomes, as we ll as increased its affinity for poly-A programmed ribosomes to the level o f native Escherichia coil tRNA(Lys). The completely unmodified anticodon st em and loop of human tRNA(CUU)(Lys1.2) With a wobble position-34 C bound AA G, but did not wobble to AAA, even when the ASL was modified with t(6)A(37) . The data suggest that tRNA(UUU)(Lys) species require anticodon domain mod ifications 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.