Wheat leaf RNA polymerases: II. Kinetic characterization and template specificities of nuclear, chloroplast, and soluble enzymes

Abstract

Further comparisons were made of DNA-dependent RNA polymerase (nucleotide triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) activities, partially purified from purified nuclear fragments and chloroplasts and from the soluble phase of young wheat leaves. All three preparations had the same cation specificities for maximal RNA polymerase activity (Mg 2+ > Mn 2+ > Ca 2+) and showed an absolute dependence on an added divalent cation. All three preparations showed the same thermal stabilities and pH optima, very similar pH-activity profiles, and the same type of kinetics with ATP as substrate. Enzyme activities showed negative cooperativity with respect to ATP concentration; the high and low K m values for ATP were not significantly different for the three preparations. The various RNA polymerase preparations differed in template specificities as follows: chloroplast DNA ⪢ nuclear DNA > calf thymus DNA > Micrococcus DNA (chloroplast enzyme); nuclear DNA ⪢ calf thymus DNA > chloroplast DNA > Micrococcus DNA (soluble enzyme); nuclear DNA > chloroplast DNA > calf thymus DNA > Micrococcus DNA (nuclear enzyme). The concentration of nuclear DNA for half-maximal RNA polymerase activity was lower for the nuclear than the soluble enzyme. Present evidence suggests that the nuclear, chloroplast, and soluble enzymes probably have identical catalytic cores but may possess different template-specifying subunits.