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)

A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease. YhaM protein has a C-terminal "HD domain," found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal "OB-fold," present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.
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PMID:Bacillus subtilis YhaM, a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria. 1239 95

1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P(1) was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P(1) fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg(2+), yielded the P(1)(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P(1)(S) fraction from E. coli K10 wild type (R(+) (1)R(+) (2)P(+)) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P(1)(S) fractions of two constitutive strains as with the P(1)(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P(1)(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn(2+) to the incubation mixtures. However, Mn(2+) inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [(32)P]CTP into RNA was overcome by Mn(2+). The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P(1)(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of beta-galactosidase by the same P(1)(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn(2+)-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.
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PMID:THE BIOSYNTHESIS OF ALKALINE PHOSPHATASE WITH A PARTICULATE FRACTION OF ESCHERICHIA COLI. 1433 60

An overlapping pathway screening (OPS) approach designed to identify and clone genes displaying parallel expression profiles as a function of induction of terminal differentiation and cellular senescence in human cells identified a novel gene old-35. Sequence and functional analysis indicates that old-35 encodes human polynucleotide phosphorylase, hPNPase(old-35). Polynucleotide phosphorylases comprise a family of phosphate dependent 3'-5' RNA exonucleases implicated in RNA regulation. Treatment of HO-1 human melanoma and additional diverse normal and tumor-derived human cell types with Type I interferon (IFN), IFN-beta or IFN-alpha, induces hPNPase(old-35) expression. To provide insights into the regulation of hPNPase(old-35), we cloned and analyzed the promoter region of this gene. These studies demonstrate that IFN-beta controls hPNPase(old-35) expression by transcriptional modulation rather than by altering mRNA stability. Transcriptional activation of hPNPase(old-35) by IFN-beta is primarily mediated by the interferon stimulatory response element (ISRE) present in its promoter. Analysis of hPNPase(old-35) expression in cell lines defective in various IFN signaling molecules confirms that hPNPase(old-35) expression is dependent upon the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Furthermore, gel shift analyses document that hPNPase(old-35) is a direct target of the interferon stimulated gene factor 3 (ISGF3) complex. The hPNPase(old-35) gene spans approximately 54 kb of genomic DNA and is distributed on 28 exons and 27 introns. hPNPase(old-35) maps to 2p15-2p16.1, a region implicated in hereditary nonpolyposis colorectal cancer, Carney complex, Doyne's honeycomb retinal dystrophy and several other diseases. To provide insights into PNPase function in vivo, we have also cloned the mouse PNPase(old-35) cDNA, mPNPase(old-35). Induction of hPNPase(old-35) by IFN treatment as well as during differentiation and senescence suggest that this gene may play a significant role in regulating cellular growth and that overlapping gene expression changes, also induced by IFN, may contribute to these important physiological processes.
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PMID:Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPase(old-35), a Type I interferon inducible early response gene. 1456 61

RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.
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PMID:Global analysis of Escherichia coli RNA degradosome function using DNA microarrays. 1498 Dec 37

Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7.0 or pH 5.0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5.0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments.
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PMID:Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance. 1513 96

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

Plastid isoprenoids (including hormones and photosynthetic pigments) are essential for plant growth and development, but relatively little is known of how the production of their metabolic precursors via the recently elucidated methylerythritol phosphate (MEP) pathway is regulated. We have identified an Arabidopsis (Arabidopsis thaliana) mutant that survives an otherwise lethal block of the MEP pathway with fosmidomycin (FSM). In rif10 (resistant to inhibition with FSM 10) plants, the accumulation of flux-controlling enzymes of the pathway is posttranscriptionally up-regulated. Strikingly, this phenotype is linked to a lower accumulation of plastidial isoprenoid pigments such as chlorophylls and carotenoids, resulting in mutant plants that are paler and smaller than the wild type. The rif10 mutant is impaired in plastid RNA processing due to a T-DNA insertion in the coding region of the At3g03710 gene encoding the chloroplast-targeted exoribonuclease polyribonucleotide phosphorylase. FSM resistance and other rif10-like phenotypes were also observed in wild-type Arabidopsis, tomato (Lycopersicon esculentum), and rice (Oryza sativa) seedlings grown in the presence of sublethal concentrations of chloramphenicol (an inhibitor of protein synthesis in plastids). By contrast, treatment with norflurazon (an inhibitor of carotenoid biosynthesis causing a similar pale cotyledon phenotype) did not result in FSM resistance. Together, the results support that plastome-encoded proteins are involved in negatively regulating the posttranscriptional accumulation of specific nuclear-encoded MEP pathway enzymes in chloroplasts. Regulation of the MEP pathway by a mechanism dependent on plastid cues might function under physiological conditions to finely adjust plastidial isoprenoid biosynthesis to the metabolic capabilities or requirements of plastids.
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PMID:Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis. 1653 78

RNase R is a processive, 3' to 5' hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3' overhang is present. Using a variety of specifically designed substrates, we show here that a 3' overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5' overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3' to 5' polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3' termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.
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PMID:Substrate recognition and catalysis by the exoribonuclease RNase R. 1689 80

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPase-deficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite- and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.
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PMID:Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii. 1735 Nov 18

Human polynucleotide phosphorylase (hPNPase(old-35)) is a type I IFN-inducible 3',5' exoribonuclease that mediates mRNA degradation. In melanoma cells, slow and sustained overexpression of hPNPase(old-35) induces G(1) cell cycle arrest ultimately culminating in apoptosis, whereas rapid overexpression of hPNPase(old-35) directly promotes apoptosis without cell cycle changes. These observations imply that inhibition of cell cycle progression and induction of apoptosis by hPNPase(old-35) involve multiple intracellular targets and signaling pathways. We now provide evidence that the apoptosis-inducing activity of hPNPase(old-35) is mediated by activation of double-stranded RNA-dependent protein kinase (PKR). Activation of PKR by hPNPase(old-35) precedes phosphorylation of eukaryotic initiation factor-2alpha and induction of growth arrest and DNA damage-inducible gene 153 (GADD153) that culminates in the shutdown of protein synthesis and apoptosis. Activation of PKR by hPNPase(old-35) also instigates down-regulation of the antiapoptotic protein Bcl-x(L). A dominant-negative inhibitor of PKR, as well as GADD153 antisense or bcl-x(L) overexpression, effectively inhibits apoptosis induction by hPNPase(old-35). These studies elucidate a novel pathway by which an evolutionary conserved RNA-metabolizing enzyme, hPNPase(old-35), regulates cell growth and viability.
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PMID:Activation of double-stranded RNA dependent protein kinase, a new pathway by which human polynucleotide phosphorylase (hPNPase(old-35)) induces apoptosis. 1780


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