title: 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.
abstract: 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.
mesh_terms: D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000235:genetics*; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012366:RNA, Transfer, Val / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
chemical_list: D004271:DNA, Fungal; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012345:RNA, Transfer, Amino Acid-Specific; D012366:RNA, Transfer, Val; D014157:Transcription Factors