Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.
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PMID:Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase. 31 25

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.
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PMID:Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site. 32 11

The synthesis of poly(mo5U) requires a high concentration (2.7 mg/ml) of polynucleotide phosphorylase as well as a long reaction time (48 h). The resulting polynucleotide has a chain length of approximately 100 nucleotides. It shows no indication of a stable secondary structure. When poly(mo5U) is mixed with poly(A), a triple-stranded complex poly(A) . 2poly(mo5U) is formed. This complex has a melting temperature of 68.5 +/- 0.5 degrees C at 150 mMNa+ and exhibits a hysteresis loop between melting and reformation of the complex having a delta Tm of 11.5 degrees C. Poly-5-methoxyuridylic acid stimulates the binding of Phe-tRNA to 70-S ribosomes but is inactive in directing poly(Phe) synthesis.
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PMID:Physical and coding properties of poly(5-methoxyuridylic) acid. 37 83

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.
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PMID:Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme. 37 31

The 6-aza analogues of toyocamycin and sangivamycin were prepared as potential cytotoxic agents. The toyocamycin analogue (4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-carbonitrile) could not be obtained directly from its O-acetylated precursor but was accessible via 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-thiocarboxamide. The identity of the nitrile was verified by its ultraviolet, infrared, and mass spectra, and by its conversion to the corresponding 3-carboxamide and thiocarboxamide when treated with water or hydrogen sulfide, respectively. Bioassay of the synthetic compounds in comparison with 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (6-azatubercidin) and 4-amino-2-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine revealed that the 3-thiocarboxamido derivative was more cytotoxic to the growth of mouse fibroblasts than 6-azatubercidin, effecting killing of 3T6 cells at less than or equal to 1 mug/ml. 4-Amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (but not its 2-ribofuranosyl isomer) was shown to act as a substrate for adenosine deaminase from calf intestinal mucosa with an apparent Km of 125 (vs. 20 for adenosine) and the corresponding 5'-diphosphate of 6-azatubercidin was polymerized by polynucleotide phosphorylase (Micrococcus luteus) in the presence of Mn2+ to afford a homopolymer and copolymers with adenosine. The copolymers directed the binding of [3H]lysyl-tRNA to the A-site of ribosomes from Escherichia coli, but could not be used for the synthesis of polylsine in a cellfree system. The copolymer consiting of adenosine and 6-azatubercidin in a 2:1 ratio was found to form a 1:1 complex with poly(uridylic acid) at 4degreesC.
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PMID:Synthesis and biological activity of pyrazolo[3,4,-d]pyrimidine nucleosides and nucleotides related to tubercidin, toyocamycin, and sangivamycin. 76 33

Polyriboadenylate polymerase was isolated from Escherichia coli PR7 (RNase I-, pnp) in good yield and high purity. The enzyme catalyzes the polymerization of ATP and ADP. These polymerizations show an initial lag which can be removed by the addition of poly(A). However, poly(A) does not function as a primer. UDP and CDP can also serve as substrates but with decreased efficiency. The polymerization of CDP is enhanced by the presence of an oligonucleotide which again does not function as a primer. Polymerization of [gamma-32P]ATP or [beta-32P]ADP result in products with no radioactivity. The product formed from [alpha-32P]ATP on hydrolysis with alkali yields labeled pAp and 2',3'-AMP; thus the enzyme synthesizes poly(A) chains de novo. During the polymerization of ATP, no burst of free ADP can be detected and the time course of phosphate release from ATP ro ADP follows very closely the kinetics of polymerization. dATP and dADP are effective inhibitors of poly(A) synthesis from either ATP or ADP. Sulfhydryl reagents inhibit only the polymerization of ATP and the inhibition is fully reversed by dithiothreitol. However, the enzyme can be protected from sulfhydryl reagents by preincubation with either ATP or ADP in the absence of Mg2+ which is required for polymerization. Studies using acrylamide gel electrophoresis indicate that the polymerization activity with either ATP or nucleoside diphosphates resides in the same protein. The enzyme catalyzes the following exchanges: 32Pi into ADP, 32Pi into ATP, and [14C] ADP into ATP in the presence of phosphate. While the enzyme catalyzes the phosphorolysis of its own product, (pAp-(Ap)nA), it fails to cleave the dephosphorylated product, (Ap(Ap)nA), or ribosomal RNA or tRNA in the presence of inorganic phosphate. The differences and similarities between poly(A) polymerase and polynucleotide phosphorylase are discussed. Based on the 32P exchange studies and other properties of poly(A) polymerase, a plausible mechanism for its action is proposed.
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PMID:Further studies on the isolation and properties of polyriboadenylate polymerase from Escherichia coli PR7 (RNase I-, pnp). 78 66

An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site.
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PMID:Location of protein S1 of Escherichia coli ribosomes at the 'A'-site of the codon binding site. Affinity labeling studies with a 3'-modified A-U-G analog. 82 27

Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA. Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure. In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated. Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature. At 20 degrees C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denaturated tRNATrp is found also to possess resistant and sensitive classes. About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20 degrees C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form. Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.
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PMID:No correlation between native and denatured forms of tRNA(Trp) form Escherichia coli and the resistant and sensitive molecules characterised by phosphorolysis. Two classes of conformation characterised by phosphorolysis in both native and denatured tRNA(Trp). 109 52

Nitrophenylated 5'-adenylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated pA-U-G. After reduction and subsequent bromoacetylation, an A-U-G analog was obtained, which could be used as an affinity label for the ribosomal A-U-G-binding site(s). After incubating the A-U-G affinity label with 70S ribosomes, 30S subunits programmed for initiation-factor-dependent fMet-tRNAMetf binding were obtained. Hence, the A-U-G analog had irreversibly reacted at the ribosomal decoding site. Initiation complexes which were formed with the labeled 30S subunits were puromycin-resistant. Furthermore, GTP hydrolysis, necessary for proper accommodation of initiator tRNA at the ribosomal donorsite, did not function in these complexes. These data indicate that immobilization of A-U-G at the decoding site of the ribosome allows factor-dependent initiator tRNA binding, but impairs accommodation at the donor site. The ribosomal protein(s) to which A-U-G was covalently bound at the decoding site were identified by polyacrylamide gel electrophoresis in the presence of urea or sarkosyl. The predominant affinity-labeled protein was found to be protein S18. Variation of the incubation conditions of the affinity-labeling reaction leads to attachment of A-U-G label to another ribosomal protein, S4, the ram gene product.
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PMID:Synthesis of a chemically reactive analog of the initiation codon: its reaction with ribosomes of Escherichia coli. 109 17

This report concerns the synthesis of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. Starting from 5-aminouridine the nucleoside 5'-phosphate was prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5'-diphosphate was prepared chemically and polymerised with polynucleotide phosphorylase. The aminouridine-containing trinucleotides were prepared by known enzymatic procedures. Besides an increase of stability in the secondary structure poly(nh25U) forms a triple-stranded complex with poly(A) and stimulates the poly(Phe) synthesis like poly(U). In contrast to U-nh25U-U, the triplet containing the 3'-terminal aminouridine does not stimulate the binding of Phe-tRNA to 70-S ribosomes. This behavior is discussed with respect to the influence of a modification on the stacking geometry of a codon and the base pairing scheme between the 5'-nucleotide of the anticodon and the 3'-nucleotide of the condon.
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PMID:Physical and coding properties of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. 110 84


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