THE FUNCTIONAL-ANALYSIS OF NONSENSE SUPPRESSORS DERIVED FROM IN-VITROENGINEERED SACCHAROMYCES-CEREVISIAE TRANSFER RNA(TRP) GENES

Nonsense suppressors derived from Saccharomyces cerevisiae tRNA(Trp) g enes have not been identified by classical genetic screens, although o ne can construct efficient amber (am) suppressors from them by making the appropriate anticodon mutation in vitro. Herein, a series of in vi tro constructed putative suppressor genes was produced to test if pre- tRNA(Trp) processing difficulties could help to explain the lack of cl assical tRNA(Trp)-based suppressors. It is clear that inefficient proc essing of introns from precursor tRNA(Trp), or inaccurate overall proc essing, may explain why some of these constructs fail to promote nonse nse suppression in vivo. However, deficient processing must be only on e of the reasons why classical tRNA(Trp)-based suppressors have not be en 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 st rains transformed with an intronless derivative of a putative tRNA(Trp ) ochre (oc) suppressor gene, wherein intron removal cannot pose a pro blem.