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

Abstract

The E3 strong promoter-active fragment harbors the tRNA pro (GGG) gene upstream of the promoterless L-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 310 (TACATT) and 335 (TTGGCA) regions which conformed to the Escherichia coli c 70 promoter. Differentiation of the 5P end of tRNA pro-GUS transcripts of pKG-E3 revealed that the true transcription initiation sites were located at positions 33, 34, and 36, 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 threefold , whereas block B and the 3P 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 vG value of 321.7 kcal mol 31. 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. ß 2002 Federation of European Micro-biological Societies. Published by Elsevier Science B.V. All rights reserved.