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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)

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.
J Mol Evol 1994 Dec
PMID:Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. 752 10

The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA. In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay. Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity. In an RNase II deletion mutant this was not observed. We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E.
FEMS Microbiol Lett 1996 Dec 15
PMID:A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E. 897 85

Repair of the 3'-terminal -CCA sequence of tRNA generally requires the action of the enzyme tRNA nucleotidyltransferase. However, in Escherichia coli in the absence of this enzyme, a decreased level of tRNA end repair continues. To ascertain the enzymes responsible for this residual repair, mutant strains were constructed lacking tRNA nucleotidyltransferase and other enzymes potentially involved in the process, poly(A) polymerase I and polynucleotide phosphorylase (PNPase). Strains lacking tRNA nucleotidyltransferase and either one of the other enzymes displayed decreased growth rates and increased levels of defective tRNA compared with the single cca mutant. Triple mutants lacking all three enzymes grew very slowly, had even more defective tRNA, and were devoid of activity incorporating AMP into tRNA-C-C. Overexpression of poly(A) polymerase I, but not PNPase, partially compensated for the absence of tRNA nucleotidyltransferase. These data show that poly(A) polymerase I and PNPase participate in the end repair process and are required to maintain functional tRNA levels when tRNA nucleotidyltransferase is absent.
J Biol Chem 1997 Dec 26
PMID:Functional overlap of tRNA nucleotidyltransferase, poly(A) polymerase I, and polynucleotide phosphorylase. 940 15

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.
J Bacteriol 1999 Dec
PMID:Protection against 3'-to-5' RNA decay in Bacillus subtilis. 1057 37

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
Mol Microbiol 1999 Dec
PMID:Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. 1059 33

Polynucleotide phosphorylase synthesis is autocontrolled at a post-transcriptional level in an RNase III-dependent mechanism. RNase III cleaves a long stem-loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase. The staggered cleavage by RNase III removes the upper part of the stem-loop structure, creating a duplex with a short 3' extension. Mutations or high temperatures, which destabilize the cleaved stem-loop, decrease expression of pnp, while mutations that stabilize the stem increase expression. We propose that the dangling 3' end of the duplex created by RNase III constitutes a target for polynucleotide phosphorylase, which binds to and degrades the upstream half of this duplex, hence inducing pnp mRNA instability. Consistent with this interpretation, a pnp mRNA starting at the downstream RNase III processing site exhibits a very low level of expression, regardless of the presence of polynucleotide phosphorylase. Moreover, using an in vitro synthesized pnp leader transcript, it is shown that polynucleotide phosphorylase is able to digest the duplex formed after RNase III cleavage.
EMBO J 2001 Dec 03
PMID:PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. 1172 20

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPase(old-35)), that is regulated predominantly by type I IFNs. The hPNPase(OLD-35) protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPase(old-35) in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPase(old-35), an IFN-inducible 3'-5' RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.
Proc Natl Acad Sci U S A 2002 Dec 24
PMID:Identification and cloning of human polynucleotide phosphorylase, hPNPase old-35, in the context of terminal differentiation and cellular senescence. 1247 48

The exoribonuclease polynucleotide phosphorylase (PNPase) has been implicated in mRNA processing and degradation in bacteria as well as in chloroplasts of higher plants. Here, we report the first comprehensive in vivo study of chloroplast PNPase function. Modulation of PNPase activity in Arabidopsis chloroplasts by a reverse genetic approach revealed that, although this enzyme is essential for efficient 3'-end processing of mRNAs, it is insufficient to mediate transcript degradation. Surprisingly, we identified PNPase as also being indispensable for 3'-end maturation of 23S rRNA transcripts. Analysis of tRNA amounts in transgenic Arabidopsis plants suggests a direct correlation of PNPase activity and tRNA levels, indicating an additional function of this exoribo nuclease in tRNA decay. Moreover, the extent of polyadenylated mRNAs in chloroplasts is negatively correlated with PNPase activity. Together, our results attribute novel functions to PNPase in the metabolism of all major classes of plastid RNAs and suggest an unexpectedly complex role for PNPase in RNA processing and decay.
EMBO J 2002 Dec 16
PMID:PNPase activity determines the efficiency of mRNA 3'-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts. 1248 11

A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40:1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor polynucleotide phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.
J Bacteriol 2003 Dec
PMID:The Streptomyces coelicolor polynucleotide phosphorylase homologue, and not the putative poly(A) polymerase, can polyadenylate RNA. 1464 89

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.
Nucleic Acids Res 2003 Dec 15
PMID:The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation. 1465 5


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