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

Published online by Cambridge University Press:  06 September 2001

MAURIZIO BRIGOTTI
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
DOMENICA CARNICELLI
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
ALESSANDRA PALLANCA
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
SIMONA RIZZI
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
PAOLA ACCORSI
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
LUCIO MONTANARO
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
SIMONETTA SPERTI
Affiliation:
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy
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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 (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.

Type
Research Article
Copyright
1999 RNA Society

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