2'-Deoxy-2'-azidoadenosine triphosphate and 2'-deoxy-2'-fluoroadenosine triphosphate as substrates and inhibitors for Escherichia coli DNA-dependent RNA polymerase

The effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AZTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.     

5'-C-methylnucleoside triphosphates in the reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase

It was shown that RNA-polymerase is able to discriminate diastereoisomers of 5'-methyl-substituted analogs of ribonucleoside triphosphates (rNTP). Under conditions of soil substrate reactions when the analog is added to the presynthesized ternary complexes, D-allo- and L-talo-stereoisomers incorporate into RNA 100 and 1000 times, respectively, less effectively, then the natural rNTP. The effectivities of incorporation of other 2'- and 3'-substituted analogs of rNTP were measured under the same conditions and compared with that for 5'-Me-rNTP. It was shown also that RNA-polymerase does not support long-chain RNA synthesis from 5'-Me-rNTP in the absence of natural rNTP. No more then two analog residues can be attached to the 3'-end of the presynthesized RNA under such conditions. Addition of one natural rNTP to this reaction mixture results in the synthesis of long alternating RNA containing D-allo-stereoisomer and natural rNTP residues. In the case of L-talo-stereoisomer RNA elongation is not inhibited, if the distance between the analog residues in the RNA chain is not shorter then five nucleotide residues. The rate of pyrophosphorolysis from the RNA of the analogs studied was the same as for the natural rNTP residues.     

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.     

Substrate specificity of CTP synthetase from Escherichia coli

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.     

Reaction of human UMP-CMP kinase with natural and analog substrates.

UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.     

Utilizations of various uridine 5'-triphosphate analogues by DNA-dependent RNA polymerases I and II purified from liver nuclei of the cherry salmon (Oncorhynchus masou)

Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).     

The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli.

Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA. These reactions were carried out by E. coli RNA polymerase on T7 DNA in the presence of rifampicin. The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined. On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds. The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not.     

Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly[d(AT).d(AT)] template

8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide. 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E. coli RNA polymerase on a poly[d(A-T)].poly[d(A-T)] template. Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP. The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions. The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions. 8-oxy-GTP slightly inhibits poly[r(A-U)] synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP. [alpha-32P] 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.     

ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein.

Poliovirus replicase- and host factor-catalyzed copying of 3'-terminal polyadenylic acid [poly(A)] of poliovirion RNA was studied. Host factor-stimulated synthesis of polyuridylic acid [poly(U)] by the replicase required ATP in addition to UTP. ATP was not required for the oligouridylic acid-primed copying of 3'-terminal poly(A) of virion RNA. GTP, CTP, and AMP-PCP (5'-adenylyl beta-gamma methylenediphosphate, an ATP analog) could not replace ATP in host factor-stimulated synthesis of poly(U). Antibodies to poliovirus genome-linked protein (VPg) specifically precipitated in vitro-synthesized poly(U) from a host factor-stimulated reaction. The poly(U) synthesized in a host factor-stimulated reaction was shown to be attached to VPg precursor polypeptide(s) via a tyrosine-phosphate bond as found in poliovirion VPg-RNA.     

Substrate properties of C′-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N-type conformation.     

Synthesis of trifluoromethyl analogs of vitamin K as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of the trifluoromethyl analogs of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) and phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone). The reduced (naphthohydroquinone) forms of the trifluoromethyl analogs of the natural vitamins had no substrate activity but were competitive inhibitors of the reaction with a Ki in the same range as the Km of the normal substrate. The oxidized form of the trifluoromethyl analogs of vitamin K also caused inhibition by a mechanism that could not be established. Under the incubation conditions utilized, fluorine was lost from the trifluoromethyl group by a process that was dithiothreitol and high pH dependent.     

Synthesis of 2',5'-oligoadenylate analogs containing an adenine acyclonucleoside and their ability to activate human RNase L

This paper described synthesis of 2',5'-oligoadenylate (2-5A) analogs containing the purine acyclonucleoside, 9-[(2'S,3'R)-2',3',4'-trihydroxybutyl]adenine (2). The ability of the analogs to activate recombinant human RNase L was evaluated using 5'-32P-r(C11U2C7)-3' as a substrate. The EC50 value (the concentration of the 2-5A required to cleave half of the RNA) of the parent 2-5A tetramer 13 was 1.0 nM, whereas those of the analog 14 incorporating 2 at the second position from the 5'-end and the analog 15 incorporating 2 at the third position from the 5'-end were 9.0 and 1.7 nM, respectively. The analogs 14 and 15 were only 9- and 1.7-fold less potent than the parent 2-5A 13 itself, in RNase L activation ability. Furthermore, the oligodeoxynucleotide containing 2 was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than the unmodified oligodeoxynucleotide. Thus, incorporation of an acyclonucleoside into 2-5A may be useful for developing an antiviral agent based on the 2-5A system.