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.

Conservation at synonymous sites was analyzed as described in ref. (57; a procedure inspired by the SSSV statistic of ref. 58 ). The procedure takes into account whether synonymous site codons are 1-, 2-, 3-, 4-or 6-fold degenerate and the differing probabilities of transitions and transversions. Briefly, for a given pair of sequences within an alignment, a codon position was defined as a synonymous site if the same amino acid was encoded in both sequences. A 'null' substitution model was defined such that the relative probability of each possible synonymous codon substitution (including substitution with itself) at such sites may be calculated by assuming that the component nucleotides evolve neutrally. Neutral evolution was modelled using a Kimura nucleotide substitution matrix with k = 3 (59) . For each sequence pair, the divergence parameter t was set so that the total expected number of nucleotide substitutions at synonymous sites under the null model was equal to the total observed number. Next, the difference between the expected number and observed number of nucleotide substitutions was calculated at each synonymous site in the pairwise comparison. The variance at each site was calculated from the expected probabilities of each possible synonymous codon substitution, assuming a multinomial distribution. Statistics were summed, at each alignment codon position, over a phylogenetic tree as described in ref. (60) . Finally the statistics were averaged over a sliding window. An approximate P-value (probability that the mean conservation in the sliding window would be as high as observed if the null model were true) was also calculated, under the assumption of a normal distribution as an approximation to the sum of many independent multinomial distributions.