共查询到20条相似文献,搜索用时 15 毫秒
1.
The effect of 8-Br-ATP and 8-oxy-ATP on RNA synthesis on calf thymus DNA and on abortive synthesis of di- and trinucleotides on promoter AI of phage T7 delta DIII DNA in the case of an incomplete set of substrates was studied. It was shown that the ATP analogs used inhibit the RNA and di- and trinucleotide synthesis. In all cases, 8-oxy-ATP was a more effective inhibitor than 8-Br-ATP. Both analogs are incapable of being the primer and they do not replace ATP in the course of abortive initiation of pppApU synthesis. 相似文献
2.
Detailed analysis of the kinetics of inhibition of E. coli RNA-polymerase-catalyzed synthesis of dinucleotide pppApU by 8-oxy-GTP and 8-Br-GTP on promoter A1 of the bacteriophage T7 delta D111 with an incomplete set of substrates was carried out. In accordance with the mathematical models obtained, we calculated quantitative parameters of binding of these nucleotide analogs to the centers whose geometry is suitable for incorporation of ATP and UTP. 8-oxy-GTP and 8-Br-GTP compete with ATP for the binding center (their steady-state dissociation constant ratios are 2.1 and 2.4, respectively, whereas the constant for ATP is 0.3 mM) but, unlike ATP, they are not incorporated into the product. 8-oxy-GTP competes also with UTP (its steady-state dissociation constant ratio is 21.6, the constant for UTP is 0.03 mM). 8-Br-GTP does not interact with the binding center of UTP. 相似文献
3.
The qualitative and quantitative characteristics of the synthesis of the short oligonucleotides by Escherichia coli RNA polymerase on A1 promoter of the bacteriophage T7 deletion mutant delta D111 DNA in the presence of the incomplete set of nucleoside triphosphates were studied. It was shown, that in conformity with the structure of A1 promoter the oligonucleotides pppApU, pppApUpC were synthesized in the presence of ATP, UTP, CTP; the oligonucleotides pApU, pApUpC-in the presence of AMP, UTP, CTP and oligonucleotides pApU, pApUpC, pApUpCpG-in the presence of AMP, UTP, CTP, GTP. The curves of di- and trinucleotide syntheses as the functions of the substrate concentrations were obtained. The analytical formulas for the rates of the coupled synthesis were derived from these curves. A kinetic scheme that is in conformity with the experimental data was proposed. This scheme includes the stage of the reversible, random and release of di- and trinucleotides from the enzyme-template complex. 相似文献
4.
5.
6.
The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly alpha 2 beta. 相似文献
7.
The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
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. 相似文献
8.
Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis. 相似文献
9.
W von der Saal P M Anderson J J Villafranca 《The Journal of biological chemistry》1985,260(28):14993-14997
CTP synthetase from Escherichia coli catalyzes exchange of 18O from the beta gamma-bridge position of [gamma-18O4] ATP into the beta-nonbridge position. This positional isotope exchange occurs in the presence of UTP and MgCl2 but in the absence of NH3. The enzyme also has an ATPase activity in the presence of UTP that occurs under conditions that are identical to those used in the positional isotope exchange experiments. These data provide evidence for the stepwise nature of the reactions catalyzed by CTP synthetase with the initial step involving phosphorylation of UTP by ATP. The relative rate of the isotope exchange reaction is approximately 3 times faster than the ATPase reaction, but the isotope exchange rate is approximately 3% of the overall rate in the presence of NH3. These results are consistent with the ATPase reaction involving attack of water on the phosphorylated intermediate (4-phospho-UTP). The positional isotope exchange reaction is independent of the UTP concentration above saturating levels of UTP demonstrating that the order of addition of substrates is UTP followed by ATP and then NH3. 相似文献
10.
CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant. 相似文献
11.
De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG. 相似文献
12.
1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [alpha-(32)P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [(3)H]CTP, [(3)H]ATP and [(3)H]GTP separately and independently of the presence of complementary ribonucleoside 5'-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg(2+) ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [alpha-(32)P]UTP showed that most of the radioactivity was recovered in (2',3')-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [(3)H]ATP, [(3)H]GTP or [(3)H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers. 相似文献
13.
The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP. The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used. De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition. A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level. 相似文献
14.
J. G. Robertson L. J. Sparvero J. J. Villafranca 《Protein science : a publication of the Protein Society》1992,1(10):1298-1307
Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues. 相似文献
15.
Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction. 相似文献
16.
ATP analogues are studied for their effect on phosphatase and ATPase activities of Na+, K+-ATPase with the aim to obtain data concerning properties and structure of sites of high and low affinity to ATP. The activating effect of nucleotides on K+-dependent phosphatase reflecting their ability to be bound with the centres of high affinity decreases in a series: ATP, N1-oxy-ATP, CTP, JTP. In the domain of high ATP concentrations, where low affinity site is saturated, ADP is a competitive inhibition of ATPase reaction with Ki of 300 microM. The analysis of N1-oxy-ATP inhibiting effect has shown that its affinity to this site is six times less than that of ADP. The absence of the inhibiting effect of CDP, JDP, GDP and UDP in concentrations up to 10 mM testifies to the fact that sites of both low and high affinity to ATP are characterized by high specificity with respect to the adenine part of the substrate molecule. 相似文献
17.
Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A 总被引:2,自引:0,他引:2
Han GS Sreenivas A Choi MG Chang YF Martin SS Baldwin EP Carman GM 《The Journal of biological chemistry》2005,280(46):38328-38336
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. 相似文献
18.
A steady state assay for the RNA polymerase initiation reaction. 总被引:15,自引:0,他引:15
A new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction. 相似文献
19.
The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined. 相似文献
20.
CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself. 相似文献