Nucleoside Triphosphate Binding Site Research Articles

Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression

An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the AcMNPV physical map. It was identified by cotransfecting Spodoptera frugiperda cultured cells with a collection of overlapping cloned DNA fragments covering the entire AcMNPV genome and a reporter gene controlled by an early, late, or very late AcMNPV promoter. Omission of the DNA fragment containing lef-1 curtailed most late and very late gene expression but not early gene expression. lef-1 was found to be an early gene transcribed as a 1.8-kb RNA in the presence of the protein synthesis inhibitor cycloheximide. The C terminus of the predicted polypeptide product, LEF-1, contained a sequence motif characteristic of nucleoside triphosphate-binding sites.

Inhibition of RNA polymerase by captan at both DNA and substrate binding sites

RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T 7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [ 14C]captan bound to the β' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [ 14C]captan was observed to bind to the β subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [ 14C]captan with the β and β' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the α and σ subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.

An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family.

The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with Mr values of 58,854, 42,299, 15,548, 18,214 and 15,108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 bp duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the PulF and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.

Affinity labeling of Avena phytochrome with ATP analogs

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5'-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys(75),Ala(25)) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome.

Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites.

The reaction catalyzed by adenosine kinase purified from human erythrocytes proceeds via a classical ordered sequential mechanism in which adenosine is the first substrate to bind to and AMP is the last product to dissociate from the enzyme. However, the interpretation of the steady-state kinetic data is complicated by the finding that while AMP acts as a classical product inhibitor at concentrations greater than 5 mM, at lower concentrations AMP can act as an apparent activator of the enzyme under certain conditions. This apparent activation by AMP is proposed to be due to AMP allowing the enzyme mechanism to proceed via an alternative reaction pathway that avoids substrate inhibition by adenosine. Quantitative studies of the protection of the enzyme afforded by adenosine against both spontaneous and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated oxidation of thiol groups yielded "protection" constants (equivalent to enzyme-adenosine dissociation constant) of 12.8 microM and 12.6 microM, respectively, values that are more than an order of magnitude greater than the dissociation constant (Kia = 0.53 microM) for the "catalytic" enzyme-adenosine complex. These results suggest that adenosine kinase has at least two adenosine binding sites, one at the catalytic center and another quite distinct site at which binding of adenosine protects the reactive thiol group(s). This "protection" site appears to be separate from the nucleoside triphosphate binding site, and it also appears to be the site that is responsible for the substrate inhibition caused by adenosine.

Maize Leaf Adenylate Kinase

Adenylate kinase (EC 2.7.4.3) from leaves of maize (Zea mays) was purified to homogeneity using (NH(4))(2)SO(4) fractionation, followed by chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75SF, and Green A dye-ligand columns. The purified enzyme had specific activity of about 1,550 micromoles ADP produced per minute per milligram protein, and the ratio of velocities of the reverse (utilization of ATP) to forward (formation of ATP) reaction was about 1.5. The M(r) value of adenylate kinase, determined by electrophoresis in dissociating conditions and by gel filtration, was 29,000 and 31,000 respectively, suggesting monomeric nature of the enzyme. Purified preparations were stable for at least 1 month at 0 to 4 degrees C. Magnesium ions were essential for activity of adenylate kinase in both directions of the reaction. Optimal rates in the forward direction were observed at the magnesium to ADP ratio of about 0.6 to 0.8. For the reverse reaction, ATP served as a substrate only when complexed with magnesium, while AMP reacted as a free species. The enzyme preferentially utilized adenine ribonucleotides in both directions of the reaction. The nucleoside triphosphate-binding site of adenylate kinase was fairly nonspecific with regard to nucleotide species. On the other hand, the primary amino group of either adenine and cytosine moieties was essential for effective binding to the nucleoside monophosphate site of the enzyme.

Identification of a nucleoside triphosphate binding site on calf thymus RNA polymerase II.

A nucleoside triphosphate binding site on calf thymus RNA polymerase II was identified by using photoaffinity analogues of adenosine 5'-triphosphate and guanosine 5'-triphosphate. Both radiolabeled 8-azidoadenosine 5'-triphosphate (8-N3ATP) and radiolabeled 8-azidoguanosine 5'-triphosphate (8-N3GTP) bound to a single polypeptide of this enzyme. This polypeptide has a molecular mass of 37 kilodaltons and an isoelectric point of 5.4. Ultraviolet (UV) irradiation was necessary for photolabeling to occur. In addition, no labeling occurred when the probe was prephotolyzed or when the enzyme was inactivated. Furthermore, photolabeling of the enzyme could be decreased by preincubation with natural substrates. To provide evidence that the radiolabeled polypeptide forms a part of the domain of the nucleoside triphosphate binding site, experiments were performed using unlabeled 8-N3ATP. Although this unlabeled analogue was not a substrate for RNA polymerase II, it photoinactivated the enzyme in the presence of UV irradiation, and it inhibited transcription elongation by the enzyme in a competitive manner in the absence of UV irradiation. As in the case with photolabeling, photoinactivation by 8-N3ATP could be decreased by natural substrates; in both cases, purine ribonucleoside triphosphates were more efficient than pyrimidine nucleoside triphosphates. Furthermore, photoinactivation was saturable at about the same concentration as the inhibition constant for 8-N3ATP. Collectively, these results provide evidence that the radiolabeled polypeptide in calf thymus RNA polymerase II is an essential component for activity and suggest that this polypeptide may be part of this enzyme's purine ribonucleoside triphosphate binding site.

Complex RNA chain elongation kinetics by wheat germ RNA polymerase II

Kinetics of RNA chain elongation catalyzed by wheat germ RNA polymerase II have been studied using various synthetic DNA templates in the presence of excess dinucleotide monophosphate primers. With single- or double-stranded homopolymer templates, the double reciprocal plots 1/(velocity) as a function of 1/(nucleotide substrate) exhibit positive, negative or no curvature. With poly(dAT) as template, the mechanism of nucleoside monophosphate incorporation into RNA is not the ping-pong kinetic mechanism which was derived for E. coli RNA polymerase (6). Noncomplementary nucleoside triphosphates inhibit RNA transcription allosterically. Cordycepin triphosphate behaves as ATP, and not only inhibits AMP incorporation but also that of UMP and GMP on appropriate templates. The reason for this complex kinetic behavior is not yet understood. Possibilities are raised that there are several nucleoside triphosphate binding sites on wheat germ RNA polymerase II, that additional nucleoside triphosphate dependent enzymatic activities are required for reaction to occur or that the Km value for incorporation of a given nucleoside monophosphate into RNA is dependent on the length of the RNA chain and/or the nucleotide sequence surrounding the complementary base on the DNA template.

Effect of sulfhydryl group modification on the activities of 5-oxo-L-prolinase.

5-Oxo-L-prolinase was isolated from rat kidney by a new procedure; highly active and apparently homogeneous enzyme was obtained in 50% yield after 1700-fold purification. The enzyme, which couples cleavage of ATP to ADP with that of 5-oxo-L-proline to L-glutamate, is uncoupled by Ca2+, Co2+, or excess Mn2+ as well as by replacement of adenosine 5'-triphosphate or of 5-oxo-L-proline by certain analogs as previously reported. The enzyme has Mr = 325,000 and is composed of 2 apparently identical subunits. It contains 27 sulfhydryl groups/monomer, 6 of which can be titrated in the native enzyme and 2 of which are required for catalysis. One of the sulfhydryl groups that can be titrated with 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide can be protected against modification by ATP or inosine 5'-triphosphate. The findings suggest that at least 1 sulfhydryl group is at or close to the nucleoside triphosphate binding site and is involved in cleavage of NTP. 5'-p-Fluorosulfonylbenzoyl adenosine and 5'-p-fluorosulfonylbenzoyl inosine interact with the sulfhydryl group involved in NTPase activity and also with another amino acid residue of the enzyme. The data provide additional strong evidence that (a) the enzyme can bind 5-oxo-L-proline in the absence of NTP, and that it can bind NTP in the absence of 5-oxo-L-proline, and (b) the L-glutamate synthesis and NTP-cleaving activities are catalyzed by the same protein.

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase.

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.

Detection of nucleoside triphosphate binding sites of two types in Escherichia coli RNA-polymerase by affinity labeling.

The affinity labeling of E. coli RNA polymerase by periodate-oxidized uridin triphosphate (o-UTP) has been carried out under the conditions of poly(dA) and poly(dT) transcription. The extent of RNA polymerase labeling proved to be 2.5 times higher under the transcription of poly(dA) as compared to poly(dT). The amount of o-UTP attached to beta beta'-subunits has been found to decrease if RNA polymerase is labeled in the transcribed complex with poly(dT). These results as well as those obtained in our previous study (1), suggest that there are two types of binding sites for nucleoside triphosphates and their analogs in E. coli RNA polymerase.

The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.

Proximity of the nucleoside monophosphate and triphosphate binding sites on deoxyribonucleic acid polymerase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProximity of the nucleoside monophosphate and triphosphate binding sites on deoxyribonucleic acid polymeraseThomas R. KrughCite this: Biochemistry 1971, 10, 13, 2594–2599Publication Date (Print):June 1, 1971Publication History Published online1 May 2002Published inissue 1 June 1971https://doi.org/10.1021/bi00789a028RIGHTS & PERMISSIONSArticle Views38Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (693 KB) Get e-Alerts Get e-Alerts