TRANSCRIPTION OF THE INT GENE OF BACTERIOPHAGE-LAMBDA - NEW RNA-POLYMERASE BINDING-SITE AND RNA START GENERATED BY INT-CONSTITUTIVE MUTATIONS

High level synthesis of the λ Int protein depends on positive regulation by the λ cII/cIII proteins, which enhance transcription from pI, a promoter close to the int gene. The intc point mutations allow high level synthesis of the Int protein even when the cII/cIII proteins are absent. In order to understand how the intc point mutations increase the constitutive level of Int protein synthesis, we have carried out a series of binding and transcription experiments in vitro using purified Escherichia coli RNA polymerase. We have studied the binding of RNA polymerase to a DNA restriction fragment that contains the pI promoter region; we find that RNA polymerase has a much greater affinity for this restriction fragment if it has an intc point mutation. We have also transcribed the restriction fragment in vitro and analyzed the RNA products on agarose/polyacrylamide gels. If the DNA has an intc point mutation, a leftward transcript of the int gene is detected; no leftward transcripts are detected when the DNA template does not have an intc point mutation. From these results we conclude that the intc point mutations have generated an active promoter site for int gene transcription. © 1979.