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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)

We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.
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PMID:Simplified methods for large scale enzymatic synthesis of oligoribonucleotides. 67 55

Poly(8-bromoadenylic acid) (poly(8-BrA)) has been synthesized by polymerization of 8-BrADP with polynucleotide phosphorylase in the presence of oligonucleotide primers. In the absence of oligonucleotides, significant (i.e. more than 1%) polymerization does not occur. Oligo(I) primer was removed selectively from the polymer with ribonuclease T1 to yield the homopolymer, poly(8-BrA). End group analysis, based on quantitative infrared measurement of the (Ip)3I-primed polymer, indicates an average degree of polymerization of about 70 residues. The primed polymers and the homopolymer appear to have similar helical structures, probably double-stranded with mutual hydrogen bonding interaction of BrA residues. Preliminary NMR observations of poly(8-BrA) with a tetrainosinic acid primer at the 5' ends of the polymer chain ((Ip)3I-(8-BrA)n) are consistent with the existence of a rigid helical structure below the melting range of the primed polymer. Above the melting range (81 degrees) the H1' coupling constants of (Ip)3I-(8-BrA)n and of polyadenylic acid (poly(A)) suggest a significantly higher population of C3' endo conformation of ribose residues in the primed polymer than in poly(A) at 81 degrees.
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PMID:Poly(8-bromoadenylic acid): synthesis and characterization of an all-syn polynucleotide. 112 40

Conversion of uridine and cytidine to their 5'-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto unknown homouridine (1-(5'-deoxy-beta-D-allofuranosyl)uracil) and homocytidine (1-(5'-deoxy-beta-D-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5'-CH2OH chain is extended by one carbon to CH2CH2OH. Homocytidine was also phosphorylated to its 6'-phosphate and 6'-pyrophosphate analogues. In addition, it was converted, via its 2,2'-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1H and 13C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homocytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5'-nucleotidase, homoCDP was a potent inhibitor of this enzyme (Ki approximately 6 microM). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.
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PMID:Pyrimidine homoribonucleosides: synthesis, solution conformation, and some biological properties. 303 11

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.
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PMID:Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives. 631 65

The following polynucleotides containing the antibiotic tubercidin (Tu; 4-amino-7-beta-D-ribofuranosylpyrrolo-[2,3-d]pyrimidine) were enzymatically synthesized by polymerization of adenosine 5'-diphosphate-tubercidin 5'-diphosphate mixtures with polynucleotide phosphorylase: poly(A2,Tu), poly(A,Tu2), and poly(Tu). The incorporation of the antibiotic was favored by the enzyme. The polymers are compared to poly(adenylic acid) [poly(A)] with respect to their structure, conformation, and ability to direct polylysine synthesis in a ribosome-dependent protein synthesis system. From physical data (thermal melting, NMR, and circular dichroism) it is concluded that tubercidin destabilizes the structure of the polynucleotide chain and that this may be due to an altered polarization of the nucleobases and their enhanced rotation around the N-glycosylic bond. Since there is an apparent correlation between thermal unfolding of the polymers and their ability to mediate polylysine synthesis, it is suggested that partial destacking of the messenger ribonucleic acid favors its binding to the ribosome and/or its ability to enhance codon-anticodon-specific protein synthesis.
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PMID:Favored incorporation of tubercidin in poly(adenylic, 7-deazadenylic acids) and their function as messenger ribonucleic acids in protein synthesis. 723 20

The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.
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PMID:The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. 900 64

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.
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PMID:Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces. 1531 61