Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli mapped by Tn10 insertion mutations.

Abstract

Transposon Tn10 insertion mutations in the oriC (replication origin) — atp (operon for the subunits of the membrane-bound ATP synthase) region of the Escherichia coli K12 chromosome were used to define promoters of the atp operon. The Tn10 insertions were first isolated and physically mapped on the specialized transducing phage λasn20 with a genome size of 23.5 MD, the insertion into which of the 6 MD Tn10 did not reduce packaging or stability. After transfer by recombination into the chromosome, a class of six Tn10 insertions was found to reduce growth yield and expression of the atp operon determined by quantitizing synthesis of the c-subunit of the ATP synthase. These six Tn10 insertions mapped within a 400 basepair segment of chromosomal DNA in front of the atpB gene. This segment contains the coding sequence for a 14KD polypeptide designated atpI. Complementation tests showed that the mutations are cis dominant and revealed that the 14 KD atpI gene product is not essential for biosynthesis and activity of the membrane bound ATP synthase. The insertions appear to block transcription into the atp genes located downstream. The insertions had different effects on atp operon expression: the ones closest to the atpB gene gave a 80%–90% decrease in c-subunit synthesis while those 150–200 basepairs further upstream only gave a 60%–70% decrease. These differential effects are taken to indicate the presence of three transcription start sites for the atp operon, a major promoter, designated atpIp, in front of the atpI gene and two minor ones, atpB1p and atpB2p, within the coding sequence atpI in front of the atpB gene. The allocation of atpIp coincides with potential transcription start signals found by DNA sequence analysis.