Identity elements in bovine tRNA(Trp) required for the specific stimulation of gelonin, a plant ribosome-inactivating protein

Ribosome-inactivating proteins (RIPs) are RNA-N-glycosidases widely present in plants that depurinate RNA in ribosomes at a specific universally conse rved 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 uniqu ely stimulated by tRNA(Trp). The active species are avian (chicken) and mam malian (beef, rat, and rabbit) tRNA(Trp), whereas yeast tRNA(Trp) is comple tely devoid of stimulating activity. In the present article, bovine and yea st tRNA(Trp) with unmodified bases were prepared by assembly of the corresp onding genes from synthetic oligonucleotides followed by PCR and T7 RNA pol ymerase transcription of the amplified products. The two synthetic tRNAs we re fully active (bovine) or inactive (yeast) as the wild-type tRNAs. Constr uction of chimeric tRNA(Trp) transcripts identified the following bovine nu cleotides 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 cooper ation between nucleotides in both arms.