Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.     

Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum.

Three, two major and one minor, distinct RNA polymerases have been isolated and partially purified from heterotrophically grown Rhodospirillum rubrum, a facultative photosynethetic bacterium. Associated with each of these three enzymes is a distinct polyadenylic acid polyemrase. All of these enzyme activities are dependent on DNA templates and are resistant to rifampicin and streptovaricin. The structural subunit composition, the response to various chemical compounds and DNA templates, and the properties of the products of these enzymes are studied in detail and compared with those of similar enzyme activities from other bacterial systems. Several unique features have been observed in the R. rubrum enzyme systems, such as an uneven incorporation of purine and pyrimidine nucleotides by the RNA polymerases, and the presence of a lag period in the polyadenylic acid polymerase activities.     

Multiple forms of DNA-dependent DNA polymerase during early development and in somatic cells of Xenopus laevis.

Four distinct DNA-dependent DNA polymerase activities (DNA polymerases I, II, III and IV according to the order of elution from a DEAE column) have been separated from extracts of unfertilized Xenopus laevis eggs. The same activities, on the basis of their chromatographic properties, template specificities and sedimentation coefficients, have been found in embryos at least until the gastrula stage. On the other hand, Xenopus kidney cells grown in culture, as well as full grown oocytes lack DNA polymerase I. These data suggest the DNA polymerase I might be a special DNA polymerase activity involved in the extremely rapid DNA synthesis which takes place during early development of X. laevis.     

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.     

Large-scale purification and subunit structure of DNA-dependent RNA polymerase II from cauliflower inflorescence

DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from cauliflower inflorescence (Brassica oleracae, var. botrytis) was highly purified by polyethyleneimine treatment on a large scale. The solubilized enzyme was partially purified by polyethyleneimine fractionation and subjected to chromatography on DEAE-Sephadex and phosphocellulose, and subsequently to sedimentation in a glycerol gradient. The specific activity (231 nmol/mg per 10 min) of this enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Analysis of the purified RNA polymerase II by polyacrylamide gel electrophoresis under non-denaturing conditions revealed a single band. The subunit composition of the enzyme was analyzed by electrophoresis under denaturing conditions. The RNA polymerase II contained subunits with molecular weights and molar ratios (in parentheses) of 180 000(1), 130 000(2), 48 000(2), 25 000(4), and 19 500(4).     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.     

DNA-dependent RNA polymerases from Artemia salina. Subunit structure of polymerase II.

RNA polymerase II from larvae of the brine shrimp, Artemia salina, was highly purified by two cycles of DEAE-cellulose chromatography followed by centrifugation through discontinuous sucrose gradients. Gradient fractions were subjected to elctrophoresis is polyacrylamide gels containing sodium dodecyl sulfate. The subunit structure of RNA polymerase II was determined by quantitative comparison of the polypeptides and enzyme activity present in each gradient fraction. The enzyme contains one copy of each of four subunits with estimated molecular weights of 170,000, 130,000, 36,000 and 24,000. The total molecular weight agrees well with the molecular weight estimated for the native enzyme by density gradient centrifugation.     

Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.     

Three distinct forms of DNA-dependent RNA polymerase III from kidney

DNA-dependent RNA polymerases were extracted from nuclei isolated from 1 kg of pig kidney and subjected to DEAE-Sephadex chromatography using a step-wise salt gradient. Fractions corresponding to RNA polymerase III were pooled and rechromatographed on a second DEAE-Sephadex column using a linear salt gradient. At least three distinct peaks, designated as IIIA, IIIB, and IIIC were resolved. These peaks exhibited α-amanitin dose response curves characteristic of RNA polymerase III. Detection of the enzyme was facilitated by assaying with either highly polymerized calf thymus DNA and spermine or with poly [d(A-T)]. The heterogeneity of this enzyme became even more pronounced after further purification. Under the same conditions, both RNA polymerases I and II were resolved at most to two subspecies. The highly heterogeneous nature of RNA polymerase III is consistent with the large number of RNA species believed to be synthesized by this enzyme class.     

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.     

Nuclear DNA-dependent RNA polymerases of Ehrlich ascites tumor cells: two discrete -amanitin-sensitive forms

DNA-dependent RNA polymerase was solubilized from nuclei of ascites tumor cells by the standard techniques of ultrasonic treatment in 0.3 M ammonium sulfate, salt fractionation, and dialysis. Three discrete forms of RNA polymerase (I, II, III) were separated on DEAE-Sephadex A-25. Forms II and III were inhibited by α-amanitin, but no form was sensitive to rifampicin. Each form was more active with Mn⁺⁺ than with Mg⁺⁺ ions, more active with denatured than with native calf thymus DNA, and differed from the others with respect to optimal concentrations of (NH₄)₂SO₄, Mn⁺⁺ ions and DNA.