THE FUNCTIONAL-ANALYSIS OF NONSENSE SUPPRESSORS DERIVED FROM IN-VITRO ENGINEERED SACCHAROMYCES-CEREVISIAE TRANSFER RNA(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.