Isolation and properties of the transcription complex of Escherichia coli RNA polymerase

Abstract

A procedure is described which permits complete separation of a transcription complex formed with template DNA, growing RNA chain and functioning RNA polymerase, from RNA polymerase molecules which have bound to DNA but not initiated RNA synthesis. The method is based on the marked stability of the transcription complex to dissociation by high concentrations of CsCl or Cs 2SO 4 which enable banding the complex after equilibrium centrifugation. With use of the newly developed procedure, affinity of Escherichia coli RNA polymerase to T7 phage DNA was found to increase during initiation of RNA synthesis but then decrease concomitant with elongation of RNA chain presumably due to migration of the enzyme to DNA sites of weak affinity. Under the conditions of maximum affinity, the transcription complex contained one core polymerase for each T7 DNA if it was isolated by centrifugation in CsCl; in contrast, 2–6 enzyme molecules remained attached on the complex when the centrifugation was carried out in Cs 2SO 4. Thus, RNA polymerases bound to different sites of transcription initiation appear to be distinguished based on the affinity of interaction. Attempts are also described to isolate the transcription complex in vivo by Cs 2SO 4 centrifugation by Brij 58-deoxycholate lysate of lysozyme-treated Escherichia coli cells. The isolated complex contained approx. 50 polymerase molecules per Escherichia coli genome as well as other unidentified proteins.