Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.     

Partial purification and characterization of a bacterial enzyme catalyzing reductive cleavage of anthracycline glycosides.

A bacterial enzyme catalyzing the NADH-dependent reductive cleavage of certain anthracycline glycosides has been partially purified. The enzyme is acidic, stable in solution and has an estimated molecular weight of 35,000. The enzyme activity is strongly inhibited by molecular oxygen but not by cyanide or EDTA. No evidence has been found for an enzyme system or associated elements of electron transport.     

Human placenta DNA primase: purification of enzyme and analysis of RNA primer synthesis.

The immunoaffinity purification of human placenta DNA primase devoid of DNA polymerase alpha activity is described. Primase consists of 52 and 59 kDa polypeptides. They form a single protein of 330 kDa under native conditions. The polypeptide structure of primase is believed to be (52 + 59)3. Primase synthesizes the oligoribonucleotides 2-9 monomers long and multimeric oligoribonucleotides of a modal length of about 10 monomers. The following model of RNA primer synthesis is proposed: 1) primase, being in free state or in complex with Pol alpha, forms a protein trimer or another structure that includes several primases; 2) primase synthesizes de novo only the oligonucleotides 2-10 monomers in length; 3) the newly synthesized oligonucleotides dissociate in solution or translocate to either Pol alpha or a neighbouring primase unit to be further elongated with the next 7-10 mononucleotides.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: I. Occurrence of thiol proteinase inhibitors in plants and purification from Vigna unguiculata subsp. cylindrica

Inhibitors of the thiol proteinase, papain (EC 3.4.22.2), were shown to be present in 11 species of 10 genera of plants. The inhibitor activity was nondialyzable, and precipitated by ammonium sulfate. Tissue cultures from a number of plant genera consisting of rapidly dividing cells contained latent papain inhibitor that could be activated upon heating. Four isoinhibitors of plant thiol proteinases from seeds of the legume Vigna unguiculata subsp. cyclindrica were purified to apparent homogeneity by acrylamide gel electrophoresis with or without sodium dodecyl sulfate. The inhibitors were present in very small amounts compared to the trypsin inhibitors and the degree of purification of the homogeneous isoinhibitors on the assumption that all were present initially in equal amounts was 15,000- to 60,000-fold. The isoinhibitors did not inhibit pepsin, bromelain, and the serine proteinases, trypsin, chymotrypsin, and subtilisin. They were specific for papain, chymopapain, and ficin but their inhibition of the proteinase, esterase, and amidase activities of the three enzymes differed.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:inositol transferase.

A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn²⁺ but not by Mg²⁺, while that in the presence of CDP-DG was activated by either Mn²⁺ or Mg²⁺. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.     

Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD alpha were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD alpha yielded a protein (rPLD alpha a, 88 kDa) together with a shorter form (rPLD alpha b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD alpha a and rPLD alpha b showed that rPLD alpha b resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLD alpha a and rPLD alpha b are recognized by a polyclonal antibody previously raised against native soybean PLD alpha. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD alpha is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD alpha b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD alpha b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD alpha a was eluted after chelating calcium ions with EDTA. The purified rPLD alpha yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD alpha constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.     

Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.     

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.     

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.     

Molecular characterization of intergenic spacer region of 5S ribosomal RNA genes in subgenus Vigna: extensive hybridization among V. unguiculata subspecies

We report the molecular analysis of the 5S ribosomal RNA intergenic spacer (IGS) region from 57 Vigna species of subgenus Vigna. Sequence analysis revealed that the 5S IGS was highly variable in length (189?C237?bp) and sequence (58% polymorphic sites). Most of the Vigna species analysed harboured a single type of 5S rRNA repeat unit, except V. unguiculata and V. reticulata that showed multiple ??intragenomic?? 5S types. The intragenomic 5S types among the six V. unguiculata subspecies were characterized by PCR-RFLP, genomic RFLP and sequencing. The 5S IGS was phylogenetically informative (comparable to ITS-1 and ITS-2 spacers) in inferring species relationships among the Vigna species analysed. However, due to the presence of multiple intragenomic 5S types and their incomplete homogenization among V. unguiculata subspecies the relationships in section Catiang could not be resolved below species level. The results presented indicate that intraspecies hybridization might have resulted in the ??horizontal transfer?? of 5S types among the V. unguiculata subspecies, while their maintenance could be due to a slow molecular drive.     

Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme.

It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC. The gene products fumarases A, B, and C have been divided into two classes. Class I contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase. Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other [Woods, S.A., Schwartzbach, S.D., & Guest, J.R. (1988) Biochim. Biophys. Acta 954, 14-26]. In this work it is shown that purified fumarase A contains a [4Fe-4S] cluster. This conclusion is based on the following observations. Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer. (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster. Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4. Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcription termination and RNA processing in the 3''-end spacer of mouse ribosomal RNA genes.

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically.     

Further purification and characterization of the succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase from rat-liver mitochondria

Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.     

The role of a conserved guanosine residue in the hammerhead-type RNA enzyme.

We have designed a hammerhead-type RNA system which consists of three RNA fragments for normal and modified complexes which contain a non-cleavable substrate with 2'-O-methylcytidine and a guanosine-to-inosine replaced enzyme. Examination of the RNA-cleaving activity and conformational properties of the complexes suggests that the 2-amino group of a conserved guanosine residue in the loop region plays an important role for maintaining both the activity and loop conformation.     

Polynucleotides. XLII1. Limited addition of 2''O-onitrobenzyl nucleotides to the 3''-end of ribooligonucleotide with polynucleotide phosphorylase.

2'-O-o-Nitrobenzyluridine, -cytidine and -adenosine were phosphorylated with phosphoryl chloride to the corresponding 5'-phosphates and led to 5'-diphosphates by the method of Moffatt and Khorana. These 2'-O-oNB-nucleoside 5'-diphosphates were incubated with a primer CpApA and polynucleotide phosphorylase in the presence of Mn2+. Tetranucleotides CpApApU, CpApApC and CpApApA were obtained after photosensitive removal of oNB groups in yields of 54-70%.