DNA-dependent RNA polymerase from the nuclei of the spleen of white rats]

DNA-dependent RNA polymerase was isolated from rat spleen cell nuclei and was identified as A and B RNA polymerases by data on DEAE- and P-cellulose ionic exchange chromatography and on concentration dependency on bivalent ions and (NH4)2SO4. Two forms of the enzyme differed from each other in the activity in RNA synthesizing system, and their activity was completely inhibited by actinomycin, DNase and RNase.     

Archaeoglobus fulgidus RNase HII in DNA replication: enzymological functions and activity regulation via metal cofactors

RNA primer removal during DNA replication is dependent on ribonucleotide- and structure-specific RNase H and FEN-1 nuclease activities. A specific RNase H involved in this reaction has long been sought. RNase HII is the only open reading frame in Archaeoglobus fulgidus genome, while multiple RNases H exist in eukaryotic cells. Data presented here show that RNase HII from A. fulgidus (aRNase HII) specifically recognizes RNA-DNA junctions and generates products suited for the FEN-1 nuclease, indicating its role in DNA replication. Biochemical characterization of aRNase HII activity in the presence of various divalent metal ions reveals a broad metal tolerance with a preference for Mg(2+) and Mn(2+). Combined mutagenesis, biochemical competitions, and metal-dependent activity assays further clarify the functions of the identified amino acid residues in substrate binding or catalysis, respectively. These experiments also reveal that Asp129 form a second-metal binding site, and thus contribute to activity attenuation.     

RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus.

The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses.     

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.     

RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe.

We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH. T2 RNase is shown to possess single-strand specificity similar to S1 nuclease. In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity. We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation. We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe. This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops. Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase. T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure. We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis. This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme. The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed.     

Use of EDTA to minimize ionic strength dependent frequency shifts in the <Superscript>1</Superscript>H NMR spectra of urine

The ¹H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). ¹H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d₄, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of protein‐only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease

The essential type of endonuclease that removes 5′ leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called ‘PRORP’, for ‘PROtein‐only RNase P’, is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high‐molecular‐weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.     

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (n_H = 2) under k_cat/K_m conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pK_a ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.     

An alkaline phosphatase-linked assay for ribonucleases

A convenient and rapid assay for ribonucleases has been developed using commerical unlabeled materials. This assay detected less than 1 ng of RNase A. The assay was also applied to RNase T₁ and micrococcal nuclease. The phosphate end groups generated at the cleavage sites of the RNA substrate were measured by incubating with excess alkaline phosphatase and determining the phosphate released. Initial reaction rates were measured and accurate units of activity established, which is not possible with most RNase assays. Commercial preparations of alkaline phosphatase from E. coli are contaminated with RNase. A procedure was described for removal of RNase from the alkaline phosphatase preparations.     

Studies on chromatin-associated nuclease from barley leaves

A nuclease associated with the chromatin of barley leaves hasbeen solubilized and purified 20 fold. The purified preparationhydrolyzes native or denatured DNA and RNA, but exhibits nophosphodiesterase or phosphomonoesterase activity. The ratiosof RNase and DNase activities remain essentially constant throughoutall the steps of purification. The two enzyme activities hadpH optimum of 7.0 and showed similar effects of phosphate, Zn++,Mg++ and other metal cations, EDTA, inhibitors, freezing andthawing, heat treatment and precipitation by protamine sulfateand streptomycin sulfate. RNA and DNA were degraded by the enzymein endonucleolytic fashion. (Received January 30, 1971; )     

Identification of a heat-labile cellular nuclease in Staphylococcus aureus with properties similar to the extracellular nuclease (EC 3.1.4.7)

Besides the well-known heat-stable extracellular staphylococcal nuclease (EC 3.1.4.7) and cell surface bound nuclease, one more nuclease, which is heat-labile, has been identified and purified on phosphorylated cellulose column and characterized. Analyses by Sephadex G-75 gel chromatography indicates that the heat-labile cellular nuclease has molecular weight of about 16,000 similar to those of extracellular and cell-surface bound nucleases. Like the heat-stable nucleases, the heat-labile enzyme acts on both DNA and RNA, is more active on heat-denatured DNA, requires Ca²⁺ ions for activity and maximum catalytic activity is observed at pH 9.8–10 and at 45°C. The results suggest that the three enzymes have properties strikingly similar to one another and therefore may be related structurally.     

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Nucleases in chloroplast of barley

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m^-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn²⁺, Ca²⁺, Zn²⁺ ions and by N-ethylmaleimide. They do not require Mg²⁺ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg²⁺ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.     

Multiple Laminin Binding Proteins in Human Fetal Heart

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln-Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non-reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn⁺⁺ ions. Immunoblotting using anti-β₁ integrin antibodies showed that 115 kDa protein is a β₁ integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.     

Assignment of paramagnetic <Superscript>15</Superscript>N-HSQC spectra by heteronuclear exchange spectroscopy

Paramagnetic metal ions in proteins provide a rich source of structural information, but the resonance assignments required to extract the information can be challenging. Here we demonstrate that paramagnetically shifted ¹⁵N-HSQC cross-peaks can be assigned using N_Z-exchange spectroscopy under conditions in which the paramagnetic form of the protein is in dynamic equilibrium with its diamagnetic form. Even slow exchange of specifically bound metal ions may be detected within the long lifetime of ¹⁵N longitudinal magnetization of large proteins at high magnetic fields. Alternatively, the exchange can be accelerated using an excess of metal ions. In the resulting exchange spectra, paramagnetic ¹⁵N resonances become visible for residues that are not directly observed in a conventional ¹⁵N-HSQC spectrum due to paramagnetic ¹H^N broadening. The experiments are illustrated by the 30 kDa lanthanide-binding ɛ186/θ complex of DNA polymerase III in the presence of sub-stoichiometric amounts of Dy³⁺ or a mixture of Dy³⁺ and La³⁺.     

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.     

Characterization of a new RNase HII and its essential amino acid residues in the archaeon <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis> reveals a regulatory C-terminus

The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 and 50°C. The activity of wild-type protein was stimulated by Mn²⁺, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1–195), retained only partial activity, while ST0519 (residues 1–196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short α-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal α-helix is likely involved in the Mn²⁺-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII.