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)

The platelet population of man and rat can be divided into two classes of about equal size on the basis of presence/absence of an acid phosphatase which acts on para-nitrophenylphosphate (a PNPase), at pH 5. The cytochemical reaction product is in the platelet cytoplasmic matrix, without apparent association with organelles or membrane systems. We could not relate differences in staining to differences in function: all cells responded the same to activation by thrombin, ADP, or collagen, in fibrinogen binding to activated platelets, by endocytosis of fluid-phase tracers, and in internalization of latex particles. With respect to possible physiological substrates for the PNP-ase, there was no reaction product from beta-glycerophosphate, AMP, ADP, ATP, GTP, CMP, IMP, cAMP, creatine phosphate, and inositol phosphates, and the enzyme was not inhibited by 40 mM lithium. There was reaction product from tyrosine phosphate suggesting that the physiological substrate for PNP-ase is tyrosine phosphate. In rat bone marrow, megakaryocytes also were of two classes, PNPase positive and PNPase negative, suggesting that different classes of platelets arise from different classes of megakaryocytes.
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PMID:Blood platelet heterogeneity: evidence for two classes of platelets in man and rat. 752 21

The ADP analogue in which the 5'-oxygen has been replaced by a methylene group can be prepared by condensing 5'-deoxy-5'-phosphonomethyladenosine with inorganic phosphate. This analogue readily polymerizes onto the primer A-A in the presence of the enzyme polynucleotide phosphorylase and either Mg2+ or Mn2+. The initial products are of the form A-A(-cA)n-cA (where "-" and "-c" stand for the normal phosphodiester linkage and the linkage in which the 5'-oxygen is replaced with the methylene group, respectively). Treatment of these with alkali yields adenosine 2'(3')-phosphate and the series (A(-cA)n-cA containing only phosphonomethylene linkages. The decamer A(-cA)8-cA interacts with two molecules of U(-U)8-U to form a triple-standard structure that has a stability similar to that exhibited by the analogous complex formed from A(-A)8-A and U(-U)8-U. This property, along with the resistance of these oligomer analogues toward nucleases that cleave phosphodiester linkages between the phosphorus and the 5'-oxygen, should provide a strong rationale for application of phosphonomethylene linkages in schemes for therapeutic drug design that use the antisense strategy.
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PMID:Synthesis and properties of adenosine oligonucleotide analogues containing methylene groups in place of phosphodiester 5'-oxygens. 839 23

GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm. This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric. There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP. This material is generated during incubation, and none is present in the initial samples. Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate. These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity. Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase. Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity. Thus, this activity is not associated with GroEL itself. The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes. Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation.
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PMID:Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL. 881 Feb 58

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.
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PMID:Polyphosphate kinase is a component of the Escherichia coli RNA degradosome. 938 62

A 320-nucleotide RNA with several characteristic features was expressed in Bacillus subtilis to study RNA processing. The RNA consisted of a 5'-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3'-proximal end of the ermC transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the pnpA gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the pnpA strain, suggesting that it was specific for PNPase. An abundant RNA with a 3' end located in the ermC coding sequence was detected in the pnpA strain but not in the wild type, indicating that this block is specific for a different 3'-to-5' exonuclease. The finding of impediments to 3'-to-5' degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
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PMID:Protection against 3'-to-5' RNA decay in Bacillus subtilis. 1057 37

Poly(formycin phosphate) and poly(laurusin phosphate) were synthesized by polymerizing formycin and laurusin 5'-diphosphate by means of E. coli polynucleotide phosphorylase. The complex formation of these polynucleotides with cyclonucleoside polynucleotides were investigated. While poly(formycin phosphate) did not form the complex with an octanucleotide of 6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil, poly(laurusin phosphate) did form a 1: 1 complex with octanucleotide of 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine in the presence of 0.15M Na ion at neutrality and 3(o). CD spectrum of this complex showed a couple of a trough at 286 nm and a peak at 262 nm. This fact suggests that the complex has a left-handed helical conformation, which is opposite to the natural double helical polynucleotides. The cause of this phenomenon was discussed in connection with the complex of cyclonucleoside oligonucleotides.
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PMID:Polynucleotides. XXVI. Complex formation of polynucleotides derived from formycin and laurusin with cyclonucleoside oligonucleotides. 1079 23

In vitro, polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade RNA in the presence of inorganic phosphate. However, because of the high in vivo concentration of inorganic phosphate in exponentially growing cells, it has been assumed that the enzyme works exclusively as an exonuclease. Here we demonstrate that, contrary to this prediction, polynucleotide phosphorylase not only synthesizes long, highly heteropolymeric tails in vivo, but also accounts for all of the observed residual polyadenylylation in poly(A) polymerase I deficient strains. In addition, the enzyme is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing cultures of wild type E. coli.
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PMID:Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli. 1103

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.
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PMID:Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli. 1132 69

The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA. Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH. These enzymes are phosphorylases that degrade RNA from the 3'-end. A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily. Here, information from the structure of PNPase is used to address two related issues. First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme. Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction.
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PMID:Running rings around RNA: a superfamily of phosphate-dependent RNases. 1179 19

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.
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PMID:Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus. 1274 47


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