Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro

Abstract

We identified two promoters for the beta-lactamase gene of plasmid pBR322. RNA isolated from bacteria containing pBR322 or RNA transcribed in vitro on pBR322 templates was hybridized to 5' end-labeled single-stranded plasmid probes (Berk, A. J., and Sharp, P. A. (1977) Cell 12, 721-732). Electrophoretic analysis of the nuclease S1 digestion products next to Maxam-Gilbert sequencing ladders closely defines the transcriptional initiation points. The natural promoter lies near the coding sequence of the beta-lactamase gene, initiating transcription at -35 bases before the ATG initiation codon, while a second promoter initiates at positions -244 and/or -245 (on the opposite side of the Eco RI site). This promoter overlaps the promoter transcribing in the opposite direction toward the tetracycline gene(s) and starts in the -10 region of that promoter. S1 mapping of procaryotic mRNA, transcribed in vivo, allows both an accurate identification of promoters and the analysis of their transcriptional regulation.