A novel factor for DNA-dependent RNA polymerase II in rat liver nuclei

The author found, in rat liver nuclei, a novel factor which exhibited a strong inhibitory effect on RNA chain initiation by various classes of RNA polymerases (I, II and III) using an exogenous DNA template.The molecular weight of this factor was estimated to be 70 K daltons, and its activity was not affected by treatment with trypsin, RNase A, lipase C, -amylase and heat. However, its activity was inactivated by a digestion of glycosidases. The molecule is shown to contain a considerable amount of sugars by physicochemical analysis. In addition, it is elucidated that the factor is not heparin which has a similar biological activity.     

Poly(A)- and primer-independent RNA polymerase of Norovirus

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3' genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.     

A simple solubilization method for loosely and tightly chromatin-bound RNA polymerase II from rat liver nuclei

Rat liver chromatin-bound RNA polymerase II could be differentially solubilized into two distinct populations, loosely and tightly bound enzymes, by a simple method. By this method the recovery of the solubilized enzyme from the chromatin fraction could be increased considerably as compared with the procedure of Yu (1). The two chromatin-bound enzymes had different properties:

1. Loosely bound enzyme was easily extractable from chromatin with relatively mild ionic condition (0.5 M NaCl); the tightly bound enzyme had to be solubilized by more drastic conditions such as sonication or nuclease treatment.
2. Loosely bound enzyme could not efficiently transcribe the chromatin template, but the tightly bound enzyme was active toward the same template. The latter enzyme is involved in the tight complex with the RNA synthesis activating factors.
3. Cycloheximide treatment in vivo suggests that the two enzymes have different turn-over rates.

Therefore, with this simple solubilization method the functionally different two chromatin-bound RNA polymerase II activities can be estimated.     

Synthesis of dinucleoside tetraphosphates by RNA polymerase B (II) from calf thymus

Highly purified RNA polymerase B (II) from calf thymus catalyses the synthesis of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires a DNA template and bivalent cations such as Mn2+ or Mg2+. It is strongly inhibited by heparin and high concentrations of alpha-amanitin but not by rifampicin. On a given template various dinucleoside tetraphosphates of different sequence are formed although the yield depends on the nature of the template.