Relationship of ornithine decarboxylase to RNA polymerase I activity

Ornithine decarboxylase activity can be rapidly elevated 50- to 100-fold by the administration of methylxanthine derivatives such as methylisobutylxanthine. This elevation occurs just prior to the increase in RNA polymerase I activity. Inhibitors of RNA synthesis and of protein synthesis suggest that any alteration in the ornithine decarboxylase response results in a similar alteration in the level of α-amanitin-insensitive RNA polymerase. Addition of a partially purified ornithine decarboxylase preparation to the RNA polymerase assay increased both the initial rate of ³H-UTP incorporation and the length of time that the polymerase assay was linear. It is suggested that ornithine decarboxylase is the labile protein that modulates the level of RNA polymerase I.     

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.     

Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.