Implication of 5′-flanking sequence elements in expression of a plant tRNALeugene

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Abstract
A comparison of 5′-flanking sequences from 68 different nuclear plant tRNA genes was analyzed to find consensus sequences. Three conserved features stood out, all of which are present in the tRNALeu gene used in this study: (1) a high proportion of A and T residues upstream of all tRNA genes; (2) a region of low duplex stability about 30–35 bp before the coding sequence, often containing a TATA-box like motif; (3) a CAA triplet in the region of the presumed transcription start. The effect of replacement of the AT-rich upstream sequences with GC-rich sequences or unrelated AT-rich sequences was tested by progressive deletions and by inserting randomly cloned sequences upstream of the tRNA gene. GC-rich 5′-flanking sequences were found to be generally incompatible with high levels of expression. The TATA-box like motifs and the CAA triplet were removed or altered by deletion or directed mutagenesis. Mutation of the CAA triplet significantly decreased expression of the tRNALeu gene, suggesting that this CAA triplet is important for transcription efficiency, but mutation or elimination of the TATA-box like motifs generally had little effect. The presence or absence of each of these features in tRNA genes from other organisms is discussed; there are clear and interesting differences between plant tRNA genes and those of yeast and mammals.