The functional analysis of nonsense suppressors derived from in vitro engineered Saccharomyces cerevisiae tRNA ^Trp genes

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA^Trp genes have not been identified by classical genetic screens, although one can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vitro constructed putative suppressor genes was produced to test if pre-tRNA^Trp processing difficulties could help to explain the lack of classical tRNA^Trp-based suppressors. It is clear that inefficient processing of introns from precursor tRNA^Trp, or inaccurate overall processing, may explain why some of these constructs fail to promote nonsense suppression in vivo. However, deficient processing must be only one of the reasons why classical tRNA^Trp-based suppressors have not been characterized, as suppression may still be extremely weak or absent in instances where the in vitro construct can lead to an accumulation of mature tRNA^Trp. Furthermore, suppression is also very weak in strains transformed with an intronless derivative of a putative tRNA^Trp ochre (oc) suppressor gene, wherein intron removal cannot pose a problem.