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We have investigated the influence of 5'-flanking sequences on the in vivo transcription activities in yeast. Since eukaryotic tRNA genes belong to multi-copy gene families monitoring of the activity of a particular tRNA gene is not possible. We therefore used two different tRNA genes from the cellular slime mould Dictyostelium discoideum which are efficiently transcribed and processed in vivo in yeast. The original 5'-flanking sequences of the two tRNA genes were replaced by random plasmid sequences. The modified tRNA genes were introduced into Saccharomyces cerevisiae and bulk tRNAs from the transformants were analyzed for the presence and the relative number of Dictyostelium tRNA gene transcripts. Substantial differences of steady-state levels of RNA transcribed were detected dependent on the 5'-flanking sequence of the tRNA gene. Minute structural changes, such as inserting two additional nucleotides in front of a tRNA gene, can lead to drastic activity changes. The efficiency of tRNA gene transcription can be conferred by sequences located more than 40 nucleotides upstream from the 5' end of the mature tRNA coding region.
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