Forms AI and AII DNA‐dependent RNA Polymerases as Components of Two Defined Pools of Polymerase Activity in Mammalian Cells

Abstract

Two species of form A (or I) RNA polymerase [EC 2.7.7.6] were identified in eukaryotic cells [rat liver] and there is evidence that this .alpha.-amanitin-insensitive activity exists as 2 discrete pools: a pool of free activity, which is identified by its ability to transcribe poly[d(A-T)] in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex (engaged) which is unaffected by inhibitors of initiation of RNA synthesis. The principles underlying and the practical application of the technique used to define the pool of free RNA polymerase activity were analyzed in considerable detail. On the basis of actinomycin titrations of poly[d(A-T)]dependent activity in isolated organelles, it is concluded that a pool of free RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form. Different techniques used to solubilize RNA polymerase activity from nucleoli give rise to different proportions of the 2 form A RNA polymerase species (AI and AII, as defined by their differential elution from phosphocellulose): whereas low-ionic-strength extraction gives rise to form AII, high-salt, sonication extracts contain predominantly the form AI enzyme. The sonication technique results in the conversion of form AII to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilization technique, it is concluded that RNA polymerase AII is the engaged form of the enzyme found in the transcription complex. Making use of the finding that the free form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity was characterized without the requirement for solubilization techniques which might result in the conversion of 1 form to another: the free species is form AI RNA polymerase. The conclusion that 2 discrete pools of form A RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.