In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters

We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcr iption extracts were prepared from mutant tobacco plants lacking PEP, the E scherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a similar to 1 kb fragment determined that the rpoB promoter is containe d in a 15-nucleotide segment (-14 to + 1) upstream of the transcription ini tiation site ( + 1), Point mutations at every nucleotide reduced transcript ion, except at the -5 position which was neutral. Critical for rpoB promote r function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), d efining it as the promoter core. The core CAT sequence is also present in t he maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (= YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furt hermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gen e, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.