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.

The WT VEEV and SINV constructs promoted RT in vitro at 2.9% and 1.6%, respectively ( Figure 6A, lanes 1 and 10) . The RT efficiencies in tissue culture cells were much higher: 7.6% for VEEV and 6.4% for SINV ( Figure 6B, lanes 1 and 10) . Substitution of the 6 nt immediately 3 0 of the UGA codon in VEEV with the tobamovirus-like RT stimulator, CAA-UUA, increased RT both in vitro (5.8%) and in tissue culture cells (10.1%; Figure 6A and B, lane 2). Derivative constructs lacking the sequences for the predicted structures were generated ( Figure 6C ). The VEEV derivative containing only 9 nt 3 0 of the UGA codon directed just 0.2% RT in vitro and 0.8% in tissue culture cells ( Figure 6A and B, lane 3), while the SINV derivative containing only 3 nt 3 0 of the UGA codon directed just 0.4% RT in vitro and 2.0% in tissue culture cells ( Figure 6A and B, lane 9). Thus the stimulatory effect of the stem-loop sequence is $10-to 14-fold for VEEV and 3-to 4-fold for SINV, depending on the assay system. This is in direct contrast to ref. 32 where no difference was found in vitro between an insert comprising just SINV UGA-CUA and an insert comprising the entire SINV nsP3+nsP4-coding sequences.