Characterization of regions of the cyanobacterial tRNA(pro) gene that affect the expression of a beta-glucuronidase reporter gene.

Abstract

The E3 strong promoter-active fragment harbors the tRNA(pro) (GGG) gene upstream of the promoterless beta-glucuronidase (GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNA(pro) coding sequence contains two regions exhibiting strong homology to blocks A and B which are the split promoter elements of eukaryotic tRNA genes. Results in this study showed that the promoter region of tRNA(pro) gene located upstream of its coding sequence and harbored the putative -10 (TACATT) and -35 (TTGGCA) regions which conformed to the Escherichia coli sigma(70) promoter. Differentiation of the 5' end of tRNA(pro)-GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions -3, -4, and -6, while the processed sites were located at position +75, +76 and +78 with respect to the first nucleotide of the tRNA(pro) coding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3' end of tRNA(pro) gene completely abolished GUS expression. However, the presence of full-length tRNA(pro) gene did not affect the GUS expression. Downstream of the tRNA(pro) coding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted DeltaG value of -21.7 kcal x mol(-1). The absence of this stem-loop structure downstream of the tRNA(pro) coding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene.