Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters

Summary Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P₀) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.     

In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida*†

SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH₄)₂SO₄ and 0.13M (NH₄)₂SO₄ respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn²⁺. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.     

DNA-dependent RNA polymerases from Drosophila melanogaster adults: Isolation and partial characterization

In preparation for the isolation and biochemical characterization of putative RNA polymerase mutants, DNA-dependent RNA polymerases of Drosophila melanogaster adults were isolated and partially characterized. Approximately 70% of the female adult RNA polymerase is located in ovaries. Multiple forms of ovarian RNA polymerases I and II are separable by DEAE-Sephadex chromatography. The two forms of RNA polymerase II differ in ammonium sulfate optima. RNA polymerase IIA is more active with double-stranded DNA as template, whereas RNA polymerase IIB transcribes single-stranded DNA most efficiently. Rechromatography of RNA polymerase IIA on DEAE-Sephadex results in the loss of ability of this form to transcribed double-stranded DNA most efficiently. Ovariectomized carcasses have two forms of RNA polymerase I and one form of RNA polymerase II and each transcribes single-stranded DNA most efficiently. As judged by gel filtration chromatography, female adult extracts have forms of RNA polymerase II that differ in molecular weight and template preference.Supported by Grants GM23456 from the NIH and 11259 from the City University Research Foundation.     

Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

Background  

The filamentous fungus Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (Theobroma cacao L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of M. perniciosa mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation.     

Inhibitors acting on Nucleic Acid Synthesis in an Oncogenic RNA Virus

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell^1–4. Several specific inhibitors of viral DNA polymerases have been found^5–7 and Spiegelman⁸ has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of actinomycin D on virus multiplication.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

   

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.     

Isolation of DNA-dependent RNA polymerase fromStreptomyces granaticolor and its binding to phage ϕ29 DNA

Partially purified DNA-dependent RNA polymerase ofStreptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits, represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage ϕ29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases fromEscherichia coli andBacillus subtilis.     

Immunological relationships between Artemia RNA polymerases and between RNA polymerases II from different eukaryotic organisms

Summary Rabbit antibodies against Artemia RNA polymerase II have been raised and utilized to study the immunological relationships between the subunits from RNA polymerases I, II and III from this organism and RNA polymerase II from other eukaryotes. We describe here for the first time the subunit structure of Artemia RNA polymerases I and III. These enzymes have 9 and 13 subunits respectively. The anti-RNA polymerase II antibodies recognize two subunits of 19.4 and 18 kDa common to the three enzymes, and another subunit of 25.6 kDa common to RNA polymerases II and III. The antibodies against Artemia RNA polymerase II also react with the subunits of high molecular weight and with subunits of around 25 and 33 kDa of RNA polymerase II from other eukaryotes (Drosophila melanogaster, Chironomus thummi, triticum (wheat) and Rattus (rat)). This interspecies relatedness is a common feature of eukaryotic RNA polymerases.Abbreviations RNAp RNA polymerase - DPT diazophenylthioether - SDS sodium dodecylsulfate     

Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations.     

Wheat DNA Primase (RNA Primer Synthesis in Vitro,Structural Studies by Photochemical Cross-Linking,and Modulation of Primase Activity by DNA Polymerases)

DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants.     

Nontemplate Polymerization of Free Nucleotides Into Genetic Elements by Thermophilic DNA Polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life.

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