Functional in vivo verification in E. coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts

Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E. coli promoter-probe vector. The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter. Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type. This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system. A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter). This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript. Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation.