Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yeast SUP4-o tRNATyr gene.

Abstract The SUP4-o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4-o genes with deletions in the 3'-flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3' deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell-free extract. Deletions that leave five or fewer consecutive T residues in the 3'-flanking sequence of SUP4-o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4-o gene is highly correlated with the relative extent of transcription termination at the 3' end of the gene. This result indicates that SUP4-o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3'-flanking sequence also significantly decrease the ability of SUP4-o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3'-flanking sequence of SUP4 plays a role in transcription factor binding.