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At least 685 records · Page 38Linked to original sources
PubMed1994

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Amino Acid Sequence↗
PubMed1996

Characterization of individual polynucleotide molecules using a membrane channel.

We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.

Base Sequence↗
PubMed2001

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E.

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.

Base Sequence↗
PubMed2003

Down-regulation of Myc as a potential target for growth arrest induced by human polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells.

Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPaseold-35), a 3',5'-exoribonuclease. We previously demonstrated that hPNPaseold-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-beta-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action.

Antineoplastic Agents↗
PubMed2004

Modulation of yersinia type three secretion system by the S1 domain of polynucleotide phosphorylase.

Both low temperatures and encounters with host phagocytes are two stresses that have been relatively well studied in many species of bacteria. Previous work has shown that the exoribonuclease polynucleotide phosphorylase (PNPase) is required for Yersiniae to grow at low temperatures. Here, we show that PNPase also enhances the ability of Yersinia pseudotuberculosis and Yersinia pestis to withstand the killing activities of murine macrophages. PNPase is required for the optimal functioning of the Yersinia type three secretion system (TTSS), an organelle that injects effector proteins directly into host cells. Unexpectedly, the effect of PNPase on the TTSS is independent of its ribonuclease activity and instead requires its S1 RNA binding domain. In contrast, catalytically inactive enzyme does not enhance the low temperature growth effect of PNPase. Surprisingly, wild-type-like TTSS functioning was restored to the pnp mutant strain by expressing just the approximately 70 amino acid S1 domains from either PNPase, RNase R, RNase II, or RpsA. Our findings suggest that PNPase plays multifaceted roles in enhancing Yersinia survival in response to stressful conditions.

Animals↗
PubMed2001

The occurence of the syn-C3' endo conformation and the distorted backbone conformations for C4'-C5' and P-O5' in oligo and polynucleotides.

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2'endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and riboguanosine residues in nucleosides and nucleotides prefer the syn-C2'endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5'-H and the N3 of the base and, a few syn-C3'endo conformations are also observed. Evidence is presented for the occurrence of the C3'endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4'-C5' and P-O5' bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3'endo conformation and the distorted backbone sugar-phosphate bonds (C4'-C5' and P-O5') as in the earlier right-handed case.

Base Pairing↗
PubMed1995

Inhibition of OH radical-induced strand break formation of poly(U) by Ru(bpy)32+ or Ru(phen)32+ attached to the polynucleotide.

Reactions of OH radicals with poly(U) (polyuridylic acid) in the presence of Ru(bpy)32+ or Ru(phen)32+ in aqueous solutions were studied. OH radicals were produced by pulse radiolysis and their reactions with ruthenium complexes were measured spectrophotometrically under conditions were the complexes are attached to the polynucleotide. The OH radical adds to either the uracil moiety or the ruthenium complexes. The ratio of the radicals produced depends only on the ratio of their rate constants and the concentrations of poly(U) and ruthenium complexes. Similar results were obtained with uridine-5'-monosphosphate, where the ruthenium complexes are not attached to the nucleotide. Surprisingly, the yield of single-strand break formation from the OH adducts of uracil in poly(U) is much smaller than that expected on the basis of the yield measured in the absence of ruthenium complexes. Possible reasons for this behaviour are discussed.

2,2'-Dipyridyl↗
PubMed1997

Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP.

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.

Bacillus subtilis↗
PubMed1974

Polynucleotides. XXII. Synthesis and properties of poly 7-deazainosinic acid.

Poly 7-deazainosinic acid has been prepared by the deamination and phosphorylation of tubercidin and the nucleoside diphosphate was polymerised using polynucleotide phosphorylase. The polymer has similar physical properties to poly(I), but has a low thermal stability in the double-stranded complex with poly(C). Poly(7-deaza I), in contrast, forms a more stable triple-stranded complex with poly(A) than 2 poly(I). poly(A), presumably due to the higher pK value.

Circular Dichroism↗
PubMed1974

Purification of polynucleotide phosphorylase by affinity chromatography and some properties of the purified enzymes.

A method is described for the preparation of p-aminophenyl oligo(dT)-Sepharose. This matrix has been used for the purification of polynucleotide phosphorylase from both E.coli and B.stearothermophilus. The effects of temperature and pH on the binding of the different enzymes to the matrix have been investigated. B.stearothermophilus isolated by affinity chromatography may be useful in selectively removing the polyA tract on the 3'-end of mRNA's.

Bacillus↗
PubMed1986

Ultraviolet-induced 8,8-adenine dehydrodimers in oligo- and polynucleotides.

Characteristic fluorescence excitation and emission is induced by either acetone-sensitized 313 nm irradiation of mixtures of 8-bromoadenosine and adenosine or 254 nm irradiation of oligo- and polynucleotides containing adenine neighbors. The acetone-sensitized reaction involves cleavage of bromine from 8-bromoadenosine with activation of C-8, leading to formation of an 8,8-adenosine dehydrodimer. Comparable fluorescence properties arise in the unsensitized photoreaction of dApdA, pdApdA, ApA, poly(dA), poly(A), poly(dA.dT), and poly(dA.U). The previously unidentified adenine ultraviolet photoproduct described by Porschke has been isolated as several variants from solutions of pdApdA and poly(dA) irradiated at 254 nm. Based upon fluorescence spectra and mass spectra, these variants are shown to contain the 8,8-adenine dehydrodimer moiety.

Acetone↗
PubMed1986

Conformational studies of (2'-5') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.

Adenine Nucleotides↗
PubMed1986

Modified polynucleotides. VII. Impaired integrity of a synthetic DNA containing the antiherpetic agent 5-isopropyl-2'-deoxyuridine.

5-Isopropyl-2'-deoxyuridine (ip5dU) was recently recognized as a clinically useful antiherpetic (HSV-1) agent. An ip5dU-containing polynucleotide, poly (dA-dT, ip5dU) was prepared to study how physical and bio-organic properties of the synthetic DNA model poly (dA-dT) would change upon partial substitution of thymidine. Synthesis was carried out with DNA polymerase enzyme and the polymers contained 7-9% of ip5dU. It proved to be less thermostable than poly (dA-dT) and the transition width was highly increased. Although it was a very efficient template for DNA polymerase enzyme, template activity for RNA synthesis was strongly reduced by the presence of ip5dU. Diminished stability against enzymic degradation, especially against single-strand-specific Nuclease S1 was also observed.

Antiviral Agents↗
PubMed1987

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.

Base Sequence↗
PubMed1989

Differential degradation of the Escherichia coli polynucleotide phosphorylase mRNA.

The transcript covering pnp, the gene encoding polynucleotide phosphorylase, is processed by RNaseIII at the 5'-upstream site of the pnp gene. In the RNaseIII-deficient strain, three species of the unprocessed transcript with different lengths could be detected. In this study, the stability of each transcript was analyzed by SI nuclease protection assay. The results show that the half-lives of the unprocessed transcripts are 8 min, whereas the half-life of the processed transcript is 1.5 min. It is also shown that the 5' segment of the unprocessed transcripts is more stable than the middle or the 3' segment.

Endoribonucleases↗
PubMed1975

The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.

Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed.

Nucleic Acid Conformation↗
PubMed1975

Further characterization of the polynucleotide phosphorylase of Micrococcus luteus.

The purification of polynucleotide phosphorylase from Micrococcus luteus by chromatography on phosphocellulose colums is described. This procedure offers several advantages over previous procedures. Previously determined molecular weights for Form-I enzyme and Form-T enzyme derived from Form-I by limited tryptic hydrolysis were confirmed as 2.7 and 2.3 times 10-5, respectively. Form-I appears homogeneous in the ultracentrifuge, but multiple active protein species are separable by polyacrylamide gel electrophoresis. The multiple species are probably the result of proteolysis. On polyacrylamide gel electrophoresis under denaturing conditions, Form-T yielded a single size of subunit of 71,000 daltons, and Form-I yielded several bands of different molecular sizes. These results differ from earlier determinations. The amino acid compositions of Form-I and Form-T are reported. Form-I contains only between 8 and 10 cysteine residues per molecule and Form-T half that many.

Amino Acids↗