RNA polymerase interaction at the promoter-operator region of the tryptophan operon of Escherichia coli and Salmonella typhimurium

Abstract

Restriction fragments from the promoter-operator region of the tryptophan operon of Escherichia coli were isolated and the location of the DNA regions which interact with RNA polymerase bound at the trp promoter was examined. RNA polymerase protects from deoxyribonuclease I digestion a region of approximately 37 base-pairs centred on the transcription initiation site. RNA polymerase also protects HincII and AluI restriction endonuclease recognition sequences located 32 to 37 and 38 to 41 base-pairs, respectively, before the transcription start-site. Although RNA polymerase in the initiation complex protects approximately the first 20 base-pairs of the transcribed portion of the operon, other studies have shown that replacement of the nucleotide sequence beyond position + 1 by a foreign sequence does not significantly affect promoter function in vivo (Bennett & Yanofsky, 1978) . Promoter function of isolated restriction fragments was evaluated by: (1) the ability to act as a template for trp messenger RNA transcription in vitro and (2) the ability of RNA polymerase to specifically protect an HpaI restriction endonuclease cleavage site present in the trp promoter-operator region and contained in each of the fragments. Fragments with termini 78 base-pairs or more before the transcription initiation site have both promoter functions. A fragment which terminates 39 base-pairs before the transcription initiation site is not efficient in either function. This places the boundary of the sequence required for trp promoter function in vitro between 39 and 78 base-pairs preceding the transcription initiation site. Findings from functional analyses with fragments from the conserved Salmonella typhimurium trp promoter-operator region (Bennett et al., 1978b) suggest that the sequences required for trp promoter function in vitro are contained within the DNA segment extending 59 basepairs before the messenger RNA start-site.