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Abstract

Transfer RNA genes require "positive" 5'-flanking sequences to direct efficient transcription. In order to delimit the modulatory sequences present in the 5'-flank of a Drosophila tRNA Val₄ gene, an extensive series of deletion mutants was constructed and end-points determined by dideoxy sequencing. The mutants were transcribed in vitro in a Drosophila Schneider II cell-free extract. Twenty nucleotides of the 5'-flank immediately adjacent to the mature tRNA coding sequence were required for transcription. Negative modulatory sequences were contained between positions -20 to -30 and -45 to -70 relative to the mature coding sequence. The -45 to -70 sequence shares homology with inhibitory sequences previously described in the 5'-flank of tRNA genes, except that this sequence was significantly larger in length. Sequences contained between positions -38 and -45 act as positive modulatory sequences which enhance the level of transcription. In addition, a Transcription Modulation Element (TME) was identified between nucleotides -33 and -38. The TME was also found in the 5'-flanking sequences of various other tRNA genes and preliminary data suggests that it enhances transcription efficiency through its position relative to the D and T control regions