DNA-dependent RNA Polymerase of the Archaebacterium Methanobacterium thermoautotrophicum

Abstract

The DNA-dependent RNA polymerase from Methanooacterium thermoautotrophicum was purified to homogeneity under the exclusion of oxygen. The subunit composition formula of the enzyme is: O: (96000)1; A: (74000)1; B: (59000)1; C: (50000)1; D: (33000)1; E: (24500)1; F: (10500)1; G: (6500)6. This results in a molecular weight of about 390000 for the complete enzyme. An incomplete particle lacking subunit O and demonstrating a five-fold lower specific activity can be isolated in addition to the complete enzyme by heparin cellulose chromatography. Maximal RNA synthesis is obtained in the presence of about 10 mM MgCl2 and 200 mM KCl at 50 or 60°C, depending on the template. As are the other archaebacterial RNA polymerases, this enzyme is insensitive to rifampicin and streptolydigin. The spacing of the heavy subunits and the existence of a fragment particle lacking the heaviest subunit are reminiscent of the halobacterial RNA polymerase.This enzyme is therefore clearly different from eubacterial RNA polymerases but it is similar to other archaebacterial RNA polymerases, thus supporting the theory of Carl Woese concerning the existence of two kingdoms of procaryotes.