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

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.