Cytoplasmic tRNAs undergo posttranscriptional 5′ and
3′ end processing in the eukaryotic nucleus, and
CCA (which forms the mature 3′ end of all tRNAs)
must be added by tRNA nucleotidyl transferase before tRNA
can be aminoacylated and utilized in translation. Eukaryotic
3′-tRNase can endonucleolytically 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 stem). This reaction proceeds despite
a wide range in length and sequence of the 3′ end
trailer, except that mature tRNA containing the 3′
terminal CCA is not a substrate for mouse 3′-tRNase
(Nashimoto, 1997, Nucleic Acids Res 25:1148–1154).
Herein, we extend this result with Drosophila
and pig 3′-tRNase, using Drosophila melanogaster
tRNAHis as substrate. Mature tRNA is thus 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 inhibitors. 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 KI
approximately two times KM for the
normal 3′-tRNase substrate. KI
increases with each nucleotide added beyond the discriminator
base, until when tRNA+CCA is used as inhibitor,
KI is approximately forty times
the substrate KM.
The 3′-tRNase can thus remain free to process precursors
with 3′ end trailers because it is barely inhibited
by tRNA+CCA, ensuring that tRNA can progress to aminoacylation.
The active site of 3′-tRNase may have evolved to
make an especially poor fit with tRNA+CCA.