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 VEEV stem-loop sequence was chosen for further analysis due to the higher RT efficiency and its greater stimulatory effect. When the 3 0 part of the stem was deleted, RT was reduced to 0.2% in vitro and 0.9% in tissue culture cells ( Figure 6A and B, lane 4) , thus demonstrating the importance of the sequence corresponding to the 3 0 component of the predicted stem, >120 nt downstream of the RT codon. To address base pairing within the predicted stem, two mutations were constructed that were predicted to disrupt Watson-Crick interactions: 3 G residues in the 5 0 part of the stem were changed to Cs or 3 C residues in the 3 0 part of the stem were changed to Gs ( Figure 6C ). In both cases, RT was drastically reduced in both in vitro and tissue culture cell assays ( Figure 6A and B, lanes 5 and 6) . However, when the two mutations were combined such that the predicted base pairings were restored, RT recovered to near the WT level ( Figure 6A and B, lane 7) . The importance of the sequence between the two halves of the stem (referred to here as the 'loop' region but without implication about internal structure) was tested by deleting all but 5 nt of its sequence. Interestingly, this resulted in substantially higher RT than the WT level in both assay systems ( Figure 6A and B, lane 8) .