Forms AI and AII DNA-dependent RNA polymerases as components of two defined pools of polymerase activity in mammalian cells.
Two species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this alpha-amanitin-insensitive activity exists as two 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. 1. The principles underlying and the practical application of the technique used to define the pool of 'free' RNA polymerase activity have been analysed 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. 2. Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two 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. It is shown that 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 solubilisation technique, it is concluded that RNA polymerase AII is the 'engaged' form of the enzyme found in the transcription complex. 3. Making use of the finding that the 'free' form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the 'free' species is shown to be form AI RNA polymerase. 4. These conclusions that two 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.
D000546:Amanitins / Q000494:pharmacology; D002466:Cell Nucleolus / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D009994:Osmolar Concentration; D012318:RNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D014465:Ultrasonics
D000546:Amanitins; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I