Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases
widely present in plants that depurinate RNA in ribosomes
at a specific universally conserved position, A4324, in
the rat 28S rRNA. A small group of RIPs (cofactor-dependent
RIPs) require ATP and tRNA to reach maximal activity on
isolated ribosomes. Among cofactor-dependent RIPs, gelonin
is specifically and uniquely stimulated by tRNATrp.
The active species are avian (chicken) and mammalian (beef,
rat, and rabbit) tRNATrp, whereas yeast tRNATrp
is completely devoid of stimulating activity. In the present
article, bovine and yeast tRNATrp with unmodified
bases were prepared by assembly of the corresponding genes
from synthetic oligonucleotides followed by PCR and T7
RNA polymerase transcription of the amplified products.
The two synthetic tRNAs were fully active (bovine) or inactive
(yeast) as the wild-type tRNAs. Construction of chimeric
tRNATrp transcripts identified the following
bovine nucleotides as recognition elements for gelonin-stimulating
activity: G26 and bp G12-C23 in the D arm and G57, A59,
and bp G51-C63 and U52-A62 in the T arm. Among single-stranded
nucleotides, A59 has a prominent role, but full expression
of the gelonin-stimulating activity requires an extensive
cooperation between nucleotides in both arms.