Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants

Two-dimensional tryptic mapping of ¹²⁵I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species.     

Purification, Characterization, and Comparison of the DNA Polymerases from Two Primate RNA Tumor Viruses

The DNA polymerase from the Mason-Pfizer monkey virus (M-PMV), an RNA tumor virus not typical type-C or type-B, has been purified a thousand-fold over the original crude viral suspension. This purified enzyme is compared to a similarly purified DNA polymerase from the primate woolly monkey virus, a type-C virus. The two enzymes have similar template specificities but differ in their requirements for optimum activity. Both DNA polymerases have a pH optimum of 7.3 in Tris buffer. M-PMV enzyme has maximum activity with 5 mM Mg(2+) and 40 mM potassium chloride, whereas the woolly monkey virus optima are 100 mM potassium chloride with 0.8 mM Mn(2+). The apparent molecular weight of the M-PMV enzyme is approximately 110,000, whereas the woolly monkey virus polymerase is approximately 70,000. The biochemical properties of these two enzymes were also compared to a similarly purified enzyme from a type-C virus from a lower mammal (Rauscher murine leukemia virus). The results show that more similarity exists between the DNA polymerases from viruses of the same type (type-C), than between the polymerases from viruses of different types but from closely related species.     

Purification of DNA-dependent RNA polymerase II from Saccharomyces cerevisiae

A rapid procedure for the purification of RNA polymerase II from Saccharomyces cerevisiae is described. Total RNA polymerase activity was solubilized from whole cells by sonication in 0.32 M (NH4)2SO4 and RNA polymerase II purified by polyethylenimine fractionation, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, DEAE-Sephadex, and phosphocellulose. The procedure may be completed in 2.5 days and the resultant enzyme is judged to be greater than 90% pure.     

Purification and partial characterization of DNA-dependent RNA polymerase from Rhodomicrobium vannielii

DNA-dependent RNA polymerase has been isolated from Rhodomicrobium vannielii. Like those from other eubacteria, the enzyme contained four subunits: beta and beta prime (Mr 155,000), alpha (Mr 38,000), and sigma (Mr 98,000). Analysis by isoelectric focussing showed that both alpha and sigma had several forms with different isoelectric pH values. The enzyme was sensitive to rifampicin (5 ng rifampicin ml-1 gave 50% inhibition) and capable of specific promoter selection with DNA from R. vannielii, calf thymus and phage T7D111.     

Purification of DNA-dependent RNA polymerase fromStreptomyces granaticolor

RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.     

Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.     

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH₄)₂SO₄ precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

The Effect of Light on the Composition of DNA-dependent RNA Polymerases of Verticillium agaricinum

Four cellular RNA polymerases have been partially purified from cultures of Verticillium agaricinum using DEAE-cellulose chromatography. Polymerases 1 and II are probably localised in the nucleolus and non-nucleolar portion of the nucleus respectively, while III and IV are probably mitochondrial in origin. DNA stimulation and dependency on the four ribonucleoside triphosphates were shown to be characteristic of all four polymerases. The products of their activity were sensitive to ribonuclease digestion and to KOH hydrolysis. The time course study of all four polymerases showed a linear increase for 1 min followed by a plateau and a rapid decrease after 5 min. Illumination of 4-day-old cultures increases the activity of RNA polymerase I, II, and III while only that of I and III is increased in 7-day cultures. There is a similar increase in the amount of carotenoids produced by 4- and 7-day-old illuminated cultures. It is tempting therefore to suggest that ribosomes or mitochondria or both may be implicated in light-induced cartogenesis.     

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.     

Purification of the DNA-dependent RNA polymerase from the myxobacterium Stigmatella aurantiaca.

The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera.