Identification of a protein factor binding to the 5'-flanking region of a tRNA gene and being involved in modulation of tRNA gene transcription in vivo in Saccharomyces cerevisiae.

Control mechanisms of tRNA gene transcription were studied in vivo in Saccharomyces cerevisiae. In order to be able to monitor in vivo transcription products of an individual tRNA gene, a 'tester gene' was used which is readily transcribed in vivo in yeast but does not cross-hybridize with any cellular yeast tRNA. A series of insertion mutants were constructed, modifying thereby the immediate and further distant 5'-flanking region of the 'tester tRNA gene'. Small linker molecules of different length and different sequence were inserted at positions -3 and -56 on the non-coding strand. Resulting tRNA gene variants were transformed into yeast cells and in vivo synthesized products were monitored by primer extension analysis. From the experimental data we suggest that a few essential nucleotides within the flanking region are able to determine the in vivo transcription activity of the 'tester tRNA gene'. Our results are rationalized on a biochemical level by protein binding assays: At least one protein binds to the 5'-flanking region of the 'tester tRNA gene' and different protein complexes are sequestered on active or less active tRNA gene variants.