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.