Comparative Effect of Heparin on RNA Synthesis of Isolated Rat-Liver Nucleoli and Purified RNA Polymerase A

Abstract

The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e. non-specific fashion. Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.