Early evolution of eukaryotic DNA-dependent RNA polymerases

Eukaryotic DNA-dependent RNA polymerases (Pol I-III) share a conserved core of 12 subunits, which is closely related to archaeal RNA polymerases. Rpb8, a subunit found in Pol I, II and III, was thought to be restricted to eukaryotes. We show here that Rpb8 closely resembles an archaeal protein called G, found only in Crenarchaea, which identifies a last missing link between the core structure of archaeal and eukaryotic RNA polymerases.     

DNA-dependent RNA polymerases from normal mouse liver

Three forms of DNA-dependent RNA polymerase from adult mouse liver are separable by DEAE-Sephadex A-25 chromatography. Two of the forms (IA and IB) are insensitive to inhibition by α-amanitin, while the third form is completely inhibited by 0.3 μg/ml of α-amanitin. The three enzyme forms are compared to the enzymes found in adult rat liver, and to the enzymes found in several other mouse tissues.     

DNA-dependent RNA polymerases isolated from yeast mitochondria

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di-t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.     

DNA-dependent RNA polymerases of the three orders of methanogens

The DNA-dependent RNA polymerases of members of the three orders of methanogens were purified and their enzymatic properties described. The enzymes consist of 7-8 polypeptides. Although these differed in molecular mass, the four heaviest components could be allied to components of the enzyme of Methanobacterium thermoautotrophicum, W by cross-reaction with antibodies directed against the denatured polypeptides of this enzyme. The antisera against native RNA polymerases isolated from representatives of the different orders, on the other hand, gave rise to serological cross-reaction between different genera but not between different families and orders. These antisera are thus useful for taxonomic purposes. The RNA polymerase of the extreme thermophile Methanothermus fervidus shows a rather low thermostability. No factors having a stabilizing influence on the enzyme could be detected.     

Effect of actinomycin D on purified DNA-dependent RNA polymerases from normal and neoplastic tissues

The effect of low concentrations of actinomycin D was investigated, using two forms of DNA-dependent RNA polymerase (A and B) purified from normal tissues and experimental tumours, in the presence either of Mn2+ or Mg2+, and homologous DNA. The A enzyme activity was strongly inhibited by the antibiotic in presence of Mg2+ and much less in presence of Mn2+. The B enzyme activity was almost suppressed in presence of both cations. The results here reported provide support that the actinomycin D induce a cellular damage of the same extent in normal and tumour tissues.     

Synthesis of two DNA-dependent RNA polymerases in yeast

Specific activities of Saccharomyces cerevisiae RNA polymerases I and II were measured in cells growing under different nutrient conditions and throughout the mitotic cell cycle. The specific activity of RNA polymerase I (possibly the ribosomal polymerase) does not vary during the yeast cell cycle. In contrast the specific activity of RNA polymerase II (messenger polymerase) increases during the first third of the cycle and thereafter declines. The independent regulation of synthesis of these two enzymes is further emphasised by observations on the response to different nutrient conditions. Shifting cells from minimal to rich medium led to enhanced RNA polymerase I activity but very little change in activity of RNA polymerase II. Furthermore the activity of RNA polymerase I varies directly with change in growth rate whereas the activity of RNA polymerase II is approximately constant over a range of growth rates. From this data it is suggested: (i) The synthesis of these two enzymes is independently regulated; (ii) RNA polymerase I is synthesised continuously throughout the cycle whereas RNA polymerase II is synthesised periodically early in the cell cycle.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Nuclear and mitochondrial DNA-dependent RNA polymerases in bovine spermatozoa

DNA-dependent RNA polymerases have been solubilized from separated head and tail fractions from normal bovine spermatozoa and from spermatozoa carrying the 'decapitated sperm defect'. When enzyme extracts from separated heads and tails were chromatographed on DEAE-Sephadex, the head fraction was resolved into 2 distinguishable peaks eluting at about 0.11 and 0.15 M-(NH4)2SO4 while the tail fraction yielded 4 distinct peaks eluting at about 0.11, 0.15, 0.255 and 0.35 M-(NH4)2SO4. Results indentical to those observed for sperm tails were obtained with extracts prepared from highly purified mitochondria from bovine or murine heart or liver. Optimization of reaction parameters and inhibitor studies with alpha-amanitin and rifampicin revealed strong similarities between eucaryotic nuclear RNA polymerases 1 and 2 and the 2 RNA polymerases associated with sperm heads. Similar experiments comparing the RNA polymerases from somatic mitochondria and sperm tails suggested the sperm tail enzymes were mitochondrial in origin.     

Animal DNA-dependent RNA polymerases. Purification and molecular structure of hen-oviduct and liver class-B RNA polymerases.

Hen ovidcut and liver class B RNA polymerases have been extensively purified and their molecular structure has been analysed. While only one enzyme B form (BIIb) was found in liver, three forms (BI, BIIa and BIIb) were resolved from oviduct. The molecular structures of the various class B RNA polymerase forms purified from hen oviduct and liver are identical to the corresponding forms previously purified from calf thymus and rat liver. At the present level of resolution the only difference lies in a slight difference in the charge of one subunit (SB2a) of enzyme form BIIa, when comparing the mammal and bird enzymes. It is unlikely that the absence of enzyme forms BI and BIIa in purified hen liver RNA polymerase B could be related to limited and specific proteolysis during the purification, since co-purification of oviduct and liver RNA polymerase B activities from a mixture of oviduct and liver nuclei does not affect the presence of either oviduct enzyme form BI or BIIa in the final purified mixture.