Animal DNA-Dependent RNA Polymerases. Purification and Molecular Structure of Hen-Oviduct and Liver Class-B RNA Polymerases

Abstract

Hen oviduct 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.In order to investigate in vitro the role of template -RNA polymerase interactions in the specificity of the transcription of a eukaryotic protein-coding gene, purified enzymes are required in addition to an appropriate DNA template and a method for evaluating the possible selective transcription (for a discussion of this problem, see [l]). At the present time there are very few experimental systems suitable for such studies. In this respect the chick oviduct system offers a model which is especially attractive for two reasons. First, because the synthesis of ovalbumin mRNA appears to be strictly dependent on the state of hormonal stimulation, and second, because complementary DNA (cDNA) to ovalbumin mRNA is available and provides a very sensitive probe which can be used for detecting the synthesis of ovalbumin mRNA sequences in vitro (for a review, see [2]). As a first step in a study aimed at analysing the molecular mechanisms which control the transcription of the ovalbumin gene, we report here methods for largescale purification of hen oviduct and liver class B RNA polymerases. For both tissues the molecular structure of the various enzyme forms present in the purified enzyme preparations has been determined and compared to the molecular structure of mammaEiizjinr. RNA polymerase or nucleosidetriphosphate : RNA nucleotidyltransferase (EC 2.7.7.6). lian class B RNA polymerases which have been previously reported [3 -61. EXPERIMENTAL PROCEDURE Materials and MethodsThe sources of the chemical and the composition of MS and P buffers were previously described [5 -81. Human y-globulins were purchased from Sigma (St. Louis, U.S.A.). Polyacrylamide gel electrophoresis under non-denaturating conditions (in the absence or in the presence of labelled [3H]amanitin) or under denaturating conditions (in the presence of sodium dodecylsulfate or urea) was carried out as previously reported [5,6,8]. RNA polymerase activity was measured as described before [5,7] in the presence of 50 mM (NH,),-SO,. One activity unit of enzyme inco...

Two Forms of RNA Polymerase B in Yeast

“…Recently, Krebs and Chambon found a unique form of enzyme B in hen liver (BIIb) and three forms in the oviduct (BI, BIIa, and BIIb) [51]. Copurification of liver and oviduct enzymes excluded the presence of a specific protease activity in liver nuclei [51].…”

“…Copurification of liver and oviduct enzymes excluded the presence of a specific protease activity in liver nuclei [51]. Therefore, if there was a limited proteolysis of liver enzymes, it could only take place during the preparation of nuclei.…”

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

“…Samples (0.3 mL) of wheat germ RNA polymerase II (specific activity, 180 units/mg) and calf thymus RNA polymerase II (specific activity, 104 units/mg) in buffer D, but with 12.5% glycerol, containing 100 mM ammonium sulfate were each layered onto 4.6-mL linear 17.5-35% glycerol gradients in 0.05 M Tris-HCl (pH 7.9)-0.1 mM Na2EDTA-0.5 mM dithiothreitol-100 mM ammonium sulfate-0.2% Nonidet-P-40 detergent. The gradients were centrifuged for 8 h in a Spinco SW 65 rotor at 0 °C as described by Krebs and Chambon (1976). Thirteen drop fractions were collected from the bottom of the tube (19-21 fractions/gradient) and assayed immediately.…”

Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos

“…5). Figure 5 also showed that the faintly staining high mol wt polypeptides discussed above in Figure 2 (17), rat livers (15), and mouse plasmacytoma (24) all resolve into three components in nondenaturing gels. RNAP II purified from animal tissues exhibits various patterns in nondenaturing gels which were not generally correlated with physiological transitions.…”