The mechanism by which heparin stimulates transcription in isolated rat liver nuclei. Polyribonucleotide elongation rates and the number of transcribing RNA polymerase molecules present

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei; it has been suggested that this process involves either the inactivation of R Nase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 °C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to α‐amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH 4 ) 2 SO 4 and Mn 2+ ions. The size of the RNA product was analysed on denaturing formamide sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and α‐amanitin which together inhibit 97% of all RNA synthesis. These results show that inactivation of R Nase cannot be an explanation of the stimulatory effect in this system. Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [ 3 H]UTP during 1‐min incubations. The [ 3 H]UMP and [ 3 H]uridine, derived from internal nucleotides and 3′‐terminal nucleosides respectively, were separated by thin‐layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of R Nase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25–35×10 3 transcribing RNA polymerase molecules per diploid genome comprising 18–20×10 3 form A and 5–15×10 3 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2–4‐fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [ 3 H]GTP in place of [ 3 H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form‐B‐mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.