Characterization of regions of the cyanobacterial tRNApro gene that affect the expression of a β-glucuronidase reporter gene

Abstract

The E3 strong promoter-active fragment harbors the tRNApro(GGG) gene upstream of the promoterless β-glucuronidase (GUS) reporter gene in plasmid pKG-E3. The 74-bp tRNAprocoding 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 tRNAprogene located upstream of its coding sequence and harbored the putative -10 (TACATT) and -35 (TTGGCA) regions which conformed to the Escherichia coli σ70promoter. Differentiation of the 5′ end of tRNApro-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 tRNAprocoding sequence. The presence of block A decreased GUS activity about three-fold, whereas block B and the 3′ end of tRNAprogene completely abolished GUS expression. However, the presence of full-length tRNAprogene did not affect the GUS expression. Downstream of the tRNAprocoding sequence in chromosomal DNA contained a 32-bp stem-loop structure with a predicted ΔG value of -21.7 kcal mol-1. The absence of this stem-loop structure downstream of the tRNAprocoding sequence in pKG-E3 resulted in read-through transcription into the adjoining GUS gene. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.