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.     

Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.     

Purification and properties of a high specific activity protein kinase from wheat germ

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on casein-Sepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per milligram of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMP-binding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K_m value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.     

tRNA ligase catalyzes the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini

The RNA ligase RtcB is conserved in all domains of life and is essential for tRNA maturation in archaea and metazoa. Here we show that bacterial and archaeal RtcB catalyze the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini. Reactions with analogues of RNA and GTP suggest a mechanism in which RtcB heals the 3'-phosphate terminus by forming a 2',3'-cyclic phosphate before joining it to the 5'-hydroxyl group of a second RNA strand. Thus, RtcB can ligate RNA cleaved by RNA endonucleases, which generate 2',3'-cyclic phosphate and then 3'-phosphate termini on one strand, and a 5'-hydroxyl terminus on another strand.     

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4-infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk- or rli- infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4-induced anticodon nuclease cleaves lysine tRNA 5' to the wobble position, yielding 2':3'-P greater than and 5'-OH termini. Polynucleotide kinase converts them into a 3'-OH and 5' P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.     

Purification and characterisation of adenosine-3',5'-phosphate-independent protein kinase from wheat germ

cAMP-independent protein kinase was isolated from the wheat germ and purified to electrophoretic homogeneity. The molecular weight of enzyme was approximately 20,000, Km for ATP was (1 +/- 0.2) x 10(-5) M. V was 215 nmol phosphate mg enzyme-1 min-1, and the isoelectric point was at pH 9.2. The enzyme promotes phosphorylation of casein and crude wheat germ ribosomes.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Purification of influenza viral complementary RNA: its genetic content and activity in wheat germ cell-free extracts.

Influenza viral complementary RNA (cRNA) was purified free from any detectable virion-type RNA (vRNA), and its genetic content and activity in wheat germ cell-free extracts were examined. After phenol-chloroform extraction of cytoplasmic fractions from infected cells, poly(A)-containing viral cRNA is found in two forms: in single-stranded RNA and associated with vRNA in partially and fully double-stranded RNA. To purify single-stranded cRNA free of these double-stranded forms, it was necessary to employ, as starting material, RNA fractions in which cRNA was predominantly single stranded. Two RNA fractions were successfully employed as starting material: polyribosomal RNA and the total cytoplasmic RNA from infected cells treated with 100 mug of cycloheximide (CM) per ml at 3 h after infection. In WSN virus-infected canine kidney (MDCK) cells, the addition of CM at 3 h after infection stimulates the production of cRNA threefold and causes a very large increase in the proportion of the cytoplasmic cRNA which is single stranded; double-stranded RNA forms are greatly reduced in amount. Total cRNA was obtained by oligo(dT)-cellulose chromatography, and single-stranded cRNA was separated from double-stranded forms by Sepharose 4B chromatography. The cRNA preparation purified from polyribosomes consists of 95% single-stranded cRNA, with the remaining 5% apparently being double-stranded RNA forms. The cRNA preparation purified from CM-treated cells (CM cRNA) is even more pure: 100% of the radiolabeled RNA is single-stranded cRNA. Annealing experiments, in which a limited amount of 32P-labeled genome RNA was annealed to the cRNA, indicate that the purified cRNA contains at least 84 to 90% of the genetic information in the vRNA genome. Purified viral cRNA (CM cRNA) is very active in directing the synthesis of virus-specific proteins in wheat germ cell-free extracts.     

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.     

Purification and properties of polynucleotide kinase of calf thymus.

Polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) has been purified approx. 1500-fold from calf thymus. This enzyme phosphorylates 5'-hydroxyl termini in DNA using ATP as phosphate donor. RNA is phosphorylated at a much lower rate than DNA. The reaction requires the presence of a divalent cation, preferably Mg2+ or Mn2+ and is sensitive to sulfhydryl antagonists. The optimum pH for enzyme activity is 5.5. Enzyme activity is inhibited by low concentrations of inorganic sulfate and by some sulfate polymers. The kinase-catalyzed incorporation of the terminal phosphate of ATP into polynucleotides is inhibited by other nucleoside and deoxynucleoside triphosphates. The enzyme molecule has a molecular weight of about 70 000 and a Stokes radius of 4.3 nm. It has a frictional ratio of 1.44 indicating an asymmetrical structure. Calf thymus tissue should provide a useful alternative source for preparation of mammalian polynucleotide kinase.     

Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.     

Purification and properties of a translation inhibitor from wheat germ.

A translation inhibitor from wheat germ has been purified more than 400-fold to apparent homogeneity. The inhibitor is a basic protein with a molecular weight of 30 000. This protein effectively blocks protein synthesis in animal cell-free extracts but does not affect protein synthesis in intact cells. Inhibition occurs at a ribosome to inhibitor molar ratio of 100:1, indicating an enzymic mechanism of action. The wheat germ protein inhibits the translation of endogenous mRNA, exogenous mRNA, and poly(uridylic acid) at a step in polypeptide chain elongation and without breakdown of the polysomes. Neither the aminoacylation reaction nor mRNA degradation is affected by the inhibitor. An interesting feature of the inhibition reaction is that it requires, in addition to the wheat germ inhibitor, both ATP and tRNA. The function of these two compounds in the inhibition is presently unknown since neither the hydrolysis of the beta,gamma-pyrophosphate bond of ATP nor a modification of the tRNA can be demonstrated during the reaction.     

A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.     

The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.     

Characterization of wheat germ lipase

Some properties of wheat germ lipase were determined with a fluorometric assay of enzymatic cleavage converting the nonfluorescent 4-methyl umbelliferone butyrate (4-MUB) to the highly fluorescent 4-methyl umbelliferone (4-MU). Optimum reaction conditions were attained at buffer pH 7·5 and temperature 30°. Lineweaver-Burk plots were linear. Relative cation combination effectiveness as reaction activators was Ca + Mg + K > Ca + Mg + K + Na > Ca + Mg + Na > Ca + Mg > Mg > Ca, with no reaction effects of K, Na, and K + Na without Ca or Mg. Highly significant inhibitors of lipase reaction were CN⁻, aflatoxin, Cu²⁺, Fe³⁺, S²⁻, and EDTA.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.