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The 5′ and 3′ extremities of the satellite tobacco necrosis virus translational enhancer domain contribute differentially to stimulation of translation

Published online by Cambridge University Press:  13 February 2002

ROSALINDE VAN LIPZIG
Affiliation:
Vakgroep Moleculaire Genetica, Universiteit Gent, B-9000 Gent, Belgium Present address: Aventis CropScience N.V., J. Plateaustraat 22, B-9000 Gent, Belgium.
ALEXANDER P. GULTYAEV
Affiliation:
Leiden Institute of Chemistry, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands Section of Theoretical Biology and Phylogenetics, Institute of Evolutionary and Ecological Sciences, Leiden University, 2311 GP Leiden, The Netherlands
CORNELIS W.A. PLEIJ
Affiliation:
Leiden Institute of Chemistry, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands
MARC VAN MONTAGU
Affiliation:
Vakgroep Moleculaire Genetica, Universiteit Gent, B-9000 Gent, Belgium
MARC CORNELISSEN
Affiliation:
Aventis CropScience N.V., B-9000 Gent, Belgium
FRANK MEULEWAETER
Affiliation:
Aventis CropScience N.V., B-9000 Gent, Belgium
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Abstract

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5′ and 3′ extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5′ border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5′ flanking sequences. At the 3′ end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3′ 14 nt of these 23 nt do not enhance translation. The 5′ and 3′ sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5′ nucleotides base-pair with the 3′ nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3′ nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.

Type
Research Article
Copyright
© 2002 RNA Society

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