Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA.

The binding sites of wheat germ RNA polymerase II were mapped on the cloned CaMV genome by observation of enzyme-linear DNA complexes by electron microscopy. Twelve sites are observed. Three of them are relatively stable in the presence of heparin and are found at positions 8-9, 21-23, and 41-44 map units on the physical map of the genome. These positions correspond to AT-rich regions of the viral genome which contain potential promoter sites. These results are discussed with reference to current information on the structure and expression of the CaMV genome.     

Comparative study of calf thymus and wheat germ RNA polymerase II: stability of initiation complexes and elongation rates.

Pure wheat germ RNA polymerase II but not calf thymus RNA polymerase II forms relatively stable binary complexes (half life time of 30 minutes at 0°C) with superhelical SV 40 DNA. On the contrary, the addition of a specific dinucleotide and a single ribotriphosphate permits the formation of highly stable complexes between both enzymes and SV 40 DNA. The elongation of RNA chains with preinitiated wheat germ enzyme only is stimulated by sarkosyl. These observations suggest that the wheat germ enzyme, as compared to that isolated from calf thymus, may contain a protein factor, a more native structure or both that permit efficient initiation and elongation of RNA chains on double stranded DNA.     

A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.     

Purification of wheat germ RNA ligase. II. Mechanism of action of wheat germ RNA ligase

The mechanism of action of purified wheat germ RNA ligase has been examined. ATP was absolutely required for the ligation of substrates containing 5'-OH or 5'-P and 2',3'-cyclic P or 2'-P termini. Ligation of 1 mol of 5'-P-2',3'-cyclic P-terminated poly(A) was accompanied by the hydrolysis of 1 mol of ATP to 1 mol each of AMP and PPi. Purified RNA ligase catalyzed an ATP-PPi exchange reaction, specific for ATP and dATP, and formed a covalent enzyme-adenylate complex that was detected by autoradiography following incubation with [alpha-32P]ATP and separation of the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein doublet with a molecular weight of approximately 110 kDa, the major product detected by silver staining, was labeled in these reactions. Isolated E-AMP complex was dissociated by the addition of ligatable poly(A), containing 5'-P-2',3'-cyclic P termini, to yield AMP and by the addition of PPi to yield ATP. The unique feature of the reactions leading to an exchange reaction between ATP and PPi and to the formation of an E-AMP complex was their marked stimulation (up to 400-fold) by the addition of RNA. This property distinguishes the wheat germ RNA ligase from other known RNA and DNA ligases which catalyze ATP-PPi exchange reactions and form E-AMP complexes in the absence of substrate. Thus, RNA appears to function in two capacities in the wheat germ system: as a cofactor, to stimulate the reaction of the enzyme with ATP, and as an authentic substrate for ligation.     

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme

We induced goat antibodies to Drosophila RNA polymerase II and rabbit antibodies to the isolated 215,000-dalton and 140,000-dalton polymerase II subunits (P215 and P140, respectively). Similarly, we induced rabbit antibodies to wheat germ RNA polymerase II and to the 220,000-dalton subunit and 140,000-dalton subunit (P220 and P140, respectively). Anti-polymerase antibodies precipitated the homologous native enzyme and inhibited its activity in vitro, while several of the anti-subunit sera did neither. The anti-Drosophila P215 serum specifically labeled RNA polymerase II fixed in situ on polytene chromosomes. We reacted the antibodies with polymerase subunits separated by sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to nitrocellulose ("protein blotting"). Each antibody to whole polymerase reacted with multiple subunits, while the anti-subunit sera each reacted specifically with the subunit employed as immunogen. The anti-subunit sera also cross-reacted with the analogous subunit from several heterologous polymerases II (from yeast, wheat germ, Drosophila, and calf thymus), demonstrating shared subunit-specific determinants in polymerase II from widely divergent organisms. The anti-polymerase sera also showed cross-reactivity with subunits of heterologous enzymes, but only in one case did the cross-reactivity involve subunits other than the two largest ones. Specifically, the goat anti-Drosophila polymerase serum displayed easily detectable cross-reactivity with four low molecular weight subunits of calf thymus polymerase II, providing a unique demonstration of antigenic relatedness of small RNA polymerase II subunits from different higher eukaryotes.