Some Properties of a DNA -Dependent RNA Polymerase from Halobacterium cutirubrum

Abstract

A DNA–protein–membrane complex, which incorporated ribonucleoside triphosphates (rNTP's) into a cold trichloracetic acid insoluble product, was isolated from an homogenate of the extremely halophilic Halobacterium cutirubrum by high speed centrifugation. After digestion with DNase and chromatography on a Sephadex column, the complex yielded an enzyme preparation which incorporated rNTP's in the presence of added DNA. Incorporation by both the complex and the enzyme preparation depended on all four rNTP's and was sensitive to DNase and RNase. Nearest-neighbor analyses showed that the product from the partially purified enzyme contained all four rNTP's in roughly equal proportion. The enzyme was inhibited by actinomycin D and acriflavin but not by rifampicin or streptovaricin. It showed no specificity for DNA templates. From Sephadex chromatography and centrifugation on glycerol gradients, its molecular weight was estimated to be roughly 300 000 – 400 000 daltons.For incorporation, the optimum Mg2+ concentration was 40 mM. The complex was active at all concentrations of KCl or NH4Cl up to saturation, but the partially purified enzyme was active only below 0.4 M. The lack of activity at higher concentrations was not due to irreversible denaturation of the enzyme but concentrated salt may have inhibited the reaction by altering the configuration of the DNA template.