Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element

Firth, Andrew E.; Wills, Norma M.; Gesteland, Raymond F.; Atkins, John F.

Sequence alignments of coding sequences containing RT stop codons were generated for a number of RNA virus taxa and the degree of conservation at synonymous sites was analyzed as described in the 'Materials and Methods' section. For an alignment of 63 VEEV, EEEV and WEEV sequences, this analysis revealed significantly enhanced conservation in a region comprising the $140 nt 3 0 -adjacent to the RT stop codon and a 9-codon sliding window size clearly resolved the conservation into two distinct peaks ( Figure 1B ). Inspection of the sequence alignment demonstrated the potential for base pairing between the sequences corresponding to these two peaks to form a stem-loop structure. In VEEV, the 5 0 -end of the 5 0 component of the stem is separated from the stop codon by an 8-9 nt 'spacer' and the 5 0 and 3 0 components of the stem are separated by a less-conserved 'loop' region (which may nonetheless contain structured elements) of 101 nt ( Figure 2 ). The predicted stem has 11-12 bp with a 1 nt asymmetric bulge in the centre of the 5 0 component and, despite the enhanced conservation, is further supported by a compensatory A:U to G:C substitution that occurs in some strains at the fourth base pair from the 'top' of the stem. In EEEV and WEEV, the predicted stem has 10 bp with a 1 nt asymmetric 5 0 bulge, and is separated from the stop codon by a 9 nt 'spacer' (Figure 2) . Again, the predicted stem is supported by a compensatory G:C to A:U substitution in the related Fort Morgan virus (FMV). High conservation was also noted for the 1-2 codons immediately 3 0 -adjacent to the 3 0 component of the predicted stem in VEEV, EEEV and WEEV.