SPECIFIC ATOMIC GROUPS AND RNA HELIX GEOMETRY IN ACCEPTOR STEM RECOGNITION BY A TRANSFER-RNA SYNTHETASE

Oligonucleotides that recapitulate the acceptor stems of tRNAs are sub strates for aminoacylation by many tRNA synthetases in vitro, even tho ugh these substrates are missing the anticodon trinucleotides of the g enetic code, In the case of tRNA(Ala) a single acceptor stem G.U base pair at position 3.70 is essential, based on experiments where the wob ble pair has been replaced by alternatives such as I.U, G.C, and A.U, among others, These experiments led to the conclusion that the minor-g roove free 2-amino group (of guanosine) of the G.U wobble pair is esse ntial for charging, Moreover, alanine-inserting tRNAs (amber suppresso rs) that replace G.U with mismatches such as G.A and C.A are partially active in vivo and can support growth of an Escherichia coli tRNA(Ala ) knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition, Herein we investigate the charging in vitro of oligonucleotide and full-lengt h tRNA substrates that contain mismatches at the position of the G.U p air, Although most of these substrates have undetectable activity, G.A and C.A variants retain some activity, which is, nevertheless, reduce d by at least 100-fold, Thus, the in vivo assays are much less sensiti ve to large changes in aminoacylation kinetic efficiency of 3.70 varia nts than is the in vitro assay system, Although these functional data do not clarify all of the details, it is now clear that specific atomi c groups are substantially more important in determining kinetic effic iency than is a helical distortion, By implication, the activity of mu tant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.