Modulation of the Phenotypic Expression of a Human Serine tRNA Gene by 5′-Flanking Sequences
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abstract
Mammalian nonsense suppressors provide a model system to investigate structural and functional aspects of mammalian tRNAs and their genes in vivo. To assess the role that extragenic flanking sequences may have on the expression of mammalian tRNA genes in vivo, deletion/substitutions ending in the 5'-flanking sequence or 3'-flanking sequence of a cloned human serine amber suppressor tRNA gene were constructed. The phenotypic expression of these mutant genes was examined by transfection in mammalian cells and by measuring the efficiency with which they were able to suppress an amber (UAG) nonsense mutation in the Escherichia coli chloramphenicol acetyl transferase (cat) gene. Deletion of the 5'-flanking region up to nucleotide position -66 with respect to the first nucleotide of the coding region had no effect on levels of nonsense suppression as compared to the wild-type gene; however, deletion to -18 led to a 12-fold reduction in suppressor activity. Deletion up to -1 did not further reduce suppression efficiency. Deletion of the 3'-flanking region up to 9 nucleotides downstream from the consecutive T residue termination site resulted in only a slight reduction in functional tRNA expression. In in vivo competition studies, the -18 deletion clone was less able to compete out the activity of a second suppressor tRNA gene than was the wild-type corresponding gene, suggesting that the upstream region plays a role in the formation of active transcription complexes in vivo. These results imply that the human serine tRNA gene contains an upstream regulatory region located between positions -66 and -18 that plays a positive role in modulating expression of this gene in vivo.
