The 3 ' end CCA of mature tRNA is an antideterminant for eukaryotic 3 '-tRNase

Cytoplasmic tRNAs undergo posttranscriptional 5' and 3' end processing in t he eukaryotic nucleus, and CCA (which forms the mature 3' end of all tRNAs) must be added by tRNA nucleotidyl transferase before tRNA can be aminoacyl ated and utilized in translation. Eukaryotic 3'-tRNase can endonucleolytica lly remove a 3' end trailer by cleaving on the 3' side of the discriminator base (the unpaired nucleotide 3' of the last base pair of the acceptor ste m). This reaction proceeds despite a wide range in length and sequence of t he 3' end trailer, except that mature tRNA containing the 3' terminal CCA i s not a substrate for mouse 3'-tRNase (Nashimoto, 1997, Nucleic Acids Res 2 5:1148-1154). Herein, we extend this result with Drosophila and pig 3'-tRNa se, using Drosophila melanogaster tRNA(His) as substrate. Mature tRNA is th us prevented from recycling through 3' end processing. We also tested a series of tRNAs ending at the discriminator base (-), with one C added (+C), two Cs added (+CC), and CCA added (+CCA) as 3'-tRNase in hibitors. Inhibition was competitive with both Drosophila and pig 3'-tRNase . The product of the 3'-tRNase reaction (-) is a good 3'-tRNase inhibitor, with a K-l approximately two times K-M for the normal 3'-tRNase substrate. K-l increases with each nucleotide added beyond the discriminator base, unt il when tRNA+CCA is used as inhibitor, K-l is approximately forty times the substrate K-M. The 3'-tRNase can thus remain free to process precursors wi th 3' end trailers because it is barely inhibited by tRNA+CCA, ensuring tha t tRNA can progress to aminoacylation. The active site of 3'-tRNase may hav e evolved to make an especially poor fit with tRNA+CCA.