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.

Although cytidine residues are under-represented at the position immediately 3 0 -adjacent to UGA (and other) terminators in eukaryotes, they are by no means absent (11, 12) . Thus we hypothesized that, at least in vivo, RT in SINV and other alphaviruses might be modulated by additional sequence elements. To test for the existence of such elements, we investigated the degree of phylogenetic conservation at synonymous sites downstream of known RT stop codons in alphavirus genomes, and then extended the analyses to other RNA viruses and selected cellular RT genes. Regions of enhanced conservation at synonymous sites are indicative of overlapping functional elements such as RNA secondary structures or primary nucleotide sequences with functions in addition to amino acid coding. In many cases, and in particular those cases where RT of a UGA codon had been previously assumed to be stimulated simply by the 3 0 -adjacent nucleotides CUA or CGG, we found considerably enhanced conservation at synonymous sites in the 3 0 -adjacent sequence, typically extending over a region of 100-200 nt 3 0 -adjacent to the stop codon. Here, we computationally and experimentally explore these conserved regions and their significance for RT.