Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA

Abstract

The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15°C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37°C. This difference is responsible for the apparent inhibition of A21 DNA transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enzyme is three times greater than that for the complex with the holoenzyme.