Identity elements in bovine tRNATrp required for the specific stimulation of gelonin, a plant ribosome-inactivating protein

Abstract

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 (cofactordependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. Among cofactor-dependent RIPs, gelonin is specifically and uniquely stimulated by tRNA Trp . The active species are avian (chicken) and mammalian (beef, rat, and rabbit) tRNA Trp , whereas yeast tRNA Trp is completely devoid of stimulating activity. In the present article, bovine and yeast tRNA Trp 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 tRNA Trp 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.

Ribosome‐inactivating proteins from plants: more than RNA N‐glycosidases?

“…Only avian (chicken) and mammalian (beef, rat, and rabbit) tRNAs Trp are active, whereas yeast tRNA Trp is completely devoid of stimulating activity. Construction of chimeric tRNA Trp transcripts identified several nucleotides in bovine tRNA Trp as recognition elements for gelonin-stimulating activity (61). The reason why only some RIPs show cofactor requirements is unknown.…”

Nucleotides U28–A42 and A37 in unmodified yeast tRNATrp as negative identity elements for bovine tryptophanyl‐tRNA synthetase

“…The choice of the source of yeast tRNAs came from the observation that 99% of brewer's yeast tRNA has an intact CCA end in contrast to baker's yeast tRNAs which in certain phases of the growth cycle show a pronounced degradation of the terminal CCA [15]. Bovine and yeast tRNA Trp transcripts were prepared as described in [5] by assemblage of the corresponding genes from synthetic oligonucleotides followed by PCR and T7 RNA polymerase transcription of the amplified products. Chimeric bovine–yeast tRNA Trp transcripts were synthesised using oligonucleotides designed according to the desired base substitutions.…”

“…The standard aminoacylation assay [5] contained, in 25 μl reaction mixtures, 100 mM Tris–HCl, pH 7.5, 0.1 mM EDTA, 10 mM ATP, 1 μCi of [ 3 H]tryptophan (30 Ci/mmol, Amersham), 0.24 μM tRNA, 90 nM bovine TrpRS (or, alternatively, 40 U of yeast aminoacyl‐tRNA synthetase) and 15 mM magnesium acetate. The excess of magnesium over ATP is required for a good acceptance of tryptophan by yeast tRNA Trp [6].…”

“…32 P‐Labelled transcripts were prepared by adding trace amounts of [α‐ 32 P]ATP to the T7 polymerase transcription reaction containing 1 mM of each rNTP [5]. The labelled transcripts were used to visualise by autoradiography the tRNA transcripts after native gel electrophoresis performed to evaluate the tertiary folding of the molecules.…”

“…During the course of our work [5] on the identity elements of animal tRNA Trp required for the specific stimulation of gelonin, the casual observation was made that, in contrast to the well known fact that wild‐type bovine and yeast tRNA Trp are both aminoacylated by the synthetase from bovine pancreas [6,7], the yeast synthetic tRNA Trp transcribed in vitro was not recognised by the bovine enzyme although efficiently charged by the aminoacyl‐tRNA synthetase from yeast. The identity elements reported for the cognate synthetases in both native yeast and animal tRNA Trp have been located in the anticodon loop and stem [8,9].…”