Nucleotide sequence determination of a strong promoter of the colicin E 1 plasmid. Analysis of restriction sites protected by RNA polymerase interactions before and after limited transcription

摘要

This paper presents the location and nucleotide sequence of a strong promoter of ColE 1. This promoter is of interest because of its greatly enhanced activity in the supercoiled state of the plasmid DNA (3) and its possible role in the maintenance of the plasmid replicon (4). This strong promoter is located at the restriction endonuclease Hae III f-h site 0.13 map units from the single EcoR 1 sIte proximal to the origin of DNA replication. The nucleotide sequence of the Hpa II l fragment of CoLE 1 which contains this promoter has been determined. Initiation of transcription at this promoter occurred at two positions. Limited transcription by omitting one of the four nucleotide triphosphates allowed transcription to proceed to the fourth (-UTP) and to the twelfth (-CTP) nucleotides respectively. This was used to probe the interaction between RNA polymerase and the CoLE 1_0.13 promoter by means of restriction cutting at the Hae III site at ?27 and the Hha I site at +17. RNA polymerase binding alone blocks restriction cutting at the Hae III site but not at the Hha I site. Limited transcription to the fourth nucleotide resulted in blocking at both sites. Transcription to the twelfth nucleotide resulted in partial cutting at the Hae III site and blocking at the Hha I site.