Mechanism of action of a yeast RNA ligase in tRNA splicing

The yeast endonuclease and ligase activities that carry out the splicing of tRNA precursors in vitro have been physically separated. The properties of a partially purified ligase fraction were examined. The ligase requires a divalent cation and a nucleoside triphosphate as cofactor. The product of ligation is a 2′-phosphomonoester, 3′,5′-phosphodiester linkage. The phosphate in the newly formed phosphodiester bond comes from the γ position of ATP, while the 2′ phosphate is derived from the RNA substrate. An adenylylated enzyme intermediate was identified by incorporation of label from α-³²P-ATP. Adenylylation was reversed by pyrophosphate, releasing ATP, whereas ligation was accompanied by release of AMP. Polynucleotide kinase and cyclic phosphodiesterase activities copurify with the adenylylated protein and may be required for the tRNA splicing reaction.     

Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).     

Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.     

Synthesis of (2'-5')(A)n from ATP. Characteristics of the reaction catalyzed by (2'-5')(A)n synthetase purified from mouse Ehrlich ascites tumor cells treated with interferon

The treatment of Ehrlich ascites tumor cells with mouse interferon increases the level of the latent enzyme (2'-5')(A)n synthetase. If activated by double-stranded RNA, this catalyzes the synthesis from ATP of a series of 2'-5'-oligoadenylates: (2'-5')(A)n where n extends from 2 to about 15. We isolated (2'-5')(A)n synthetase in a homogeneous state. In the presence of double-stranded RNA, the purified enzyme can convert the large majority (about 97%) of the ATP into (2'-5')(A)n and pyrophosphate, although it does not cleave the pyrophosphate. The stoichiometry of the reaction can be formulated as: (n + I) ATP leads to (2'-5') pppA(pA)n + n pyrophosphate. Added pyrophosphate does not inhibit the synthesis of (2'-5')(A)n. The extent of the reverse reaction, i.e. the pyrophosphorolysis of (2'-5')(A)n, was below the level of detection under our conditions. The affinity of the enzyme for ATP is low: the rate of the reaction increases by about 10% when the concentration of ATP is increased from 5 mM to 10 mM. The optimal concentration of double-stranded RNA increases with the concentration of the enzyme. As tested at 0.4, 2, and 10 micrograms/ml of enzyme concentrations, close to maximal (2'-5')(A)n synthesis can be obtained if reovirus double-stranded RNA or poly(I) . poly(C) are used at about half the concentration (in w/v) of the enzyme. The plot of the reaction rate versus enzyme concentration is sigmoidal. It remains to be seen if this reflects on a cooperative behavior of the enzyme.     

Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.     

The stereochemical course of nucleotidyl transfer catalysed by NAD pyrophosphorylase

NAD pyrophosphorylase catalyses nucleotidyl transfer from adenosine (R)-5'-[alpha-17O]triphosphate to nicotinamide mononucleotide with inversion of configuration at the alpha-P giving (S)-[17O]NAD+. The simplest interpretation of this observation is that the adenylyl group is transferred directly from ATP to the co-substrate by an 'in line' mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD+ regio-specifically at the adenylyl terminus of the pyrophosphate bond.     

Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase

The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate.     

Chemical and enzymatic synthesis of 4'-thio-beta-D-arabinofuranosylcytosine monophosphate and triphosphate

N4-Acetyl-1-(2, 3-di-O-acetyl-4-thio-beta-D-arabinofuranosyl) cytosine (2) was synthesized in three steps from 1-(4-thio-beta-D-arabinofuranosyl) cytosine (1). The reaction of this partially blocked 4'-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5'-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4'-thio-ara-C-5'-triphosphate 5. This triphosphate 5 was converted to 4'-thio-ara-C -5'-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules.

Under the conditions that RNA ligase converts the tetranucleotide, pA-A2-A, to larger polynucleotides, no such polymerization can be detected with the derivative, pA-A2-A(MeOEt), that possesses a terminal 2'-0-(alpha-methoxyethyl) group. The protection against self condensation offered by the methoxyethyl group in this system allows the specific joining of donor and acceptor oligonucleotides in reaction mixtures containing equimolar concentrations of the two species. Thus, the enzyme, together with ATP, converts equimolar quantities of A-A2-A and pA-A2-A(MeOEt) to A-A6-A(MeOEt) in 55% yield, while a similar reaction with A-A2-A and pU-U2-U(MeOEt) results in a 40% yield of A-A3-U3-U(MeOEt). The intermediate in these ligations is a disubstituted pyrophosphate composed of the donor molecule and the adenylate moiety deriving from ATP. In the case of the intermediate arising from the blocked adenosine tetranucleotide, the assigned structure, A5'pp5'A-A2-A(MeOEt), has been confirmed by chemical synthesis. The pyrophosphate derivative is able to participate in joining reactions in the absence of ATP. These observations constitute an efficient approach to the synthesis of larger polynucleotides from a specific series of oligonucleotide blocks since (i), the methoxyethyl group can be easily introduced into each oligonucleotide using the single addition reaction catalyzed by polynucleotide phosphorylase in the presence of a 2'-0-(alpha-methoxyethyl)nucleoside 5'-diphosphate, and (ii), the blocking group may be readily removed under mild conditions after each successive ligation reaction. Two other octanucleotides, I-I2-A-U3-U and U-U2-C-I3-A, have also been synthesized by this method, and these molecules correspond (with I substituting for G) to sequences appearing near the 3' terminus of the 6S RNA transcribed from phage lambda DNA. The terminal 3'-phosphate group serves equally well as a blocking group for specific ligation reactions in that the ligase converts equimolar amounts of A-A2-A and pA-A2-Ap to A-A6-Ap in 50% yield.     

Studies of the Rous sarcoma virus RNA: characterization of the 5'-terminus

The 5′ terminus of the Rous Sarcoma Viral 30-40S RNA was characterized as follows: Unlabeled RNA was treated with polynucleotide kinase and (γ-³²P) ATP. Degradation of the 5′-(³²P) RNA with alkali yielded labeled pAp while degradation with venom phosphodiesterase yielded labeled 5′-AMP. Dephosphorylation with alkaline phosphatase was unnecessary for the RNA to accept³²P indicating the presence of 5′-OH ends. This establishes that the base at the 5′ end of Rous Sarcoma Viral 30-40S RNA is adenine.     

Cyanamide mediated syntheses under plausible primitive earth conditions

When an aqueous solution (pH 7.0) of 3H deoxythymidine 5'-triphosphate, deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide, cyanamide and ammonium chloride was dried and heated at 60 degrees C for 18 h, oligomers were obtained in a yield of approximately 80%. After the chemical degradation of any pyrophosphate bonds present in these oligomers, linear polynucleotides of up to 7-8 units in length were isolated by DEAE cellulose column chromatography and identified by enzymatic digestion procedures. The di- and trinucleotide fractions were degraded 87% and 100% by snake venom phosphodiesterase and 39% and 9% by spleen phosphodiesterase. This synthesis of deoxythymidine oligonucleotides was conducted under potentially prebiotic conditions and may offer a possible method for the synthesis of deoxyoligonucleotides on the primitive Earth.     

Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.

Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.     

Regulation of heart muscle pyruvate dehydrogenase kinase

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).     

Positional isotope exchange analysis of the pantothenate synthetase reaction

Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PP(i) must be free to dissociate from the active site upon formation of the pantoyl-adenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.     

Protein phosphorylation by inorganic pyrophosphate in yeast mitochondria.

Inorganic pyrophosphate can function as phosphate donor in protein phosphorylation reactions in yeast mitochondria. It was shown that, when PPi substitutes for ATP as inhibitor of the pyruvate dehydrogenase reaction, maximal activity is reached after a lag-period of 30-60 minutes. 32P-labeling of peptides shows that [32P]PPi gives about 25% of the labeling obtained by [gamma-32P]ATP in the protein kinase reaction. The PPi dependent phosphorylation is increased several fold by the presence of cold ATP.     

Intermediate products of the RNA-ligase reaction]

The first and the third steps of the RNA-ligase reaction were studied. It was shown that the first step of the reaction consists in a formation of an enzyme-adenylate complex. The optimal conditions for this formation were established. Effects of acids, alkali, hydroxylamine and snake venom phosphodiesterase on the complex suggest that the linkage between the protein and adenylic acid may be of a phosphoester or phosphoamide type. Using synthetic adenylic acid pyrophosphates and mononucleotides (oligonucleotides) the RNA-ligase reaction was shown to involve intermediate pyrophosphates. It was found that the simplest pyrophosphates capable to bind to oligonucleotides in the absence of ATP are adenylic acid pyrophosphates, both of ribo- and deoxyribomononucleotides. The RNA-ligase reaction may be used for elongation of oligonucleotides by one definite mononucleotide or for incorporation of the label into the 3'-end of the polynucleotide chain.