Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eukaryotic RNA exosomes participate in 3' to 5'-processing and degradation of RNA in the nucleus and cytoplasm. RNA exosomes are multisubunit complexes composed of at least nine distinct proteins that form the exosome core. Although the eukaryotic exosome core shares structural and sequence similarity to phosphorolytic archaeal exosomes and bacterial PNPase, the eukaryotic exosome core has diverged from its archaeal and bacterial cousins and appears devoid of phosphorolytic activity. In yeast, the processive hydrolytic 3' to 5'-exoribonuclease Rrp44 associates with exosomes in the nucleus and cytoplasm. Although human Rrp44 appears homologous to yeast Rrp44, it has not yet been shown to associate with human exosomes. In the nucleus, eukaryotic exosomes interact with Rrp6, a distributive hydrolytic 3' to 5'-exoribonuclease. To facilitate analysis of eukaryotic RNA exosomes, we will describe procedures used to clone, express, purify, and reconstitute the nine-subunit human exosome and nine-, ten-, and eleven-subunit yeast exosomes. We will also discuss procedures to assess exoribonuclease activity for reconstituted exosomes.
 ...
PMID:Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae cloning, expression, purification, and activity assays. 1911 Nov 77

The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.
 ...
PMID:Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited. 1913 86

Co-immunopurification is a classical technique in which antiserum raised against a specific protein is used to purify a multiprotein complex. We describe work from our laboratory in which co-immunopurification was used to characterize the RNA degradosome of Escherichia coli, a multiprotein complex involved in RNA processing and mRNA degradation. Polyclonal rabbit antibodies raised against either RNase E or PNPase, two RNA degrading enzymes in the RNA degradosome, were used in co-immunopurification experiments aimed at studying the assembly of the RNA degradosome and mapping protein-protein interactions within the complex. In E. coli, this method has been largely supplanted by approaches in which proteins are engineered to contain tags that interact with commercially available antibodies. Nevertheless, we believe that the method described here is valid for the study of bacteria in which the genetic engineering needed to introduce tagged proteins is difficult or nonexistent. As an example, we briefly discuss ongoing work in our laboratory on the characterization of RNase E in the psychrotolerant bacterium Pseudoalteromonas haloplanktis.
 ...
PMID:Co-immunopurification of multiprotein complexes containing RNA-degrading enzymes. 1916 38

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli, it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components: the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. The variable presence of additional proteins, however, suggests that the degradosome is a flexible machine that may vary its composition in response to different conditions. Direct analysis of large protein complexes, together with simplified purification procedures, can facilitate qualitative and quantitative identification of RNA degradosome components under different physiologic and genetic conditions and can help to explain their role in the bacterial cell (see also Chapters 4, 11, 19, 20 and 22 regarding methods for the studying the degradosome and other multiprotein complexes in this volume. Herewith we describe the application of multidimensional protein identification technology (MudPIT) in the rapid and quantitative identification of RNA degradosome components. RNA degradosome preparations obtained from specific conditions are enzymatically digested. The resulting peptides are fractionated using two-dimensional (ion-exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. Bioinformatic analysis with the SEQUEST algorithm, which correlates experimentally obtained mass spectra with those predicted from peptide sequences in proteomic and translated genomic databases, allows identification of the corresponding proteins that compose the complex. The protein constituents of two or more degradosome samples are then compared to obtain a rapid evaluation of qualitative and quantitative differences in protein composition. Quantitative analysis is based on the observation that changes in relative protein abundance among different samples are reflected by statistical parameters (score values) assigned to each protein component of the RNA degradosome identified by the MudPIT approach. This correlation can be validated by independent methods such as Western blotting and determination of enzymatic activities. This fully automated procedure may be applied to the characterization of any complex protein mixture.
 ...
PMID:A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli. 1916 40

Mitochondria and chloroplasts were originally acquired by eukaryotic cells through endosymbiotic events and retain their own gene expression machinery. One hallmark of gene regulation in these two organelles is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels. This review focuses on their mechanisms of RNA degradation, and therefore mainly on the polyadenylation-stimulated degradation pathway. Overall, mitochondria and chloroplasts have retained the prokaryotic RNA decay system, despite evolution in the number and character of the enzymes involved. However, several significant differences exist, of which the presence of stable poly(A) tails, and the location of PNPase in the intermembrane space in animal mitochondria, are perhaps the most remarkable. The known and predicted proteins taking part in polyadenylation-stimulated degradation pathways are described, both in chloroplasts and four mitochondrial types: plant, yeast, trypanosome, and animal.
 ...
PMID:RNA polyadenylation and decay in mitochondria and chloroplasts. 1921 78

RNase E is an essential enzyme that catalyses RNA processing. Microdomains which mediate interactions between RNase E and other members of the degradosome have been defined. To further elucidate the role of these microdomains in molecular interactions, we studied RNase E from Vibrio angustum S14. Protein sequence analysis revealed that its C-terminal half is less conserved and structured than its N-terminal half. Within this structural disorder, however, exist five small regions of predicted structural propensity. Four are similar to interaction-mediating microdomains identified in other RNase E proteins; the fifth did not correspond to any known functional motif. The function of the V. angustum S14 enolase-binding microdomain was confirmed using bacterial two-hybrid analysis, demonstrating the conserved function of this microdomain for the first time in a species other than Escherichia coli. Further, PNPase in V. angustum S14 was shown to interact with the last 80 amino acids of the C-terminal region of RNase E. This raises the possibility that PNPase interacts with the small ordered region at residues 1026-1041. The role of RNase E as a hub protein and the implications of microdomain-mediated interactions in relation to specificity and function are discussed.
 ...
PMID:Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners. 1934 89

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3'-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.
 ...
PMID:Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance. 1986 55

OxyS is one of at least three small non-coding RNAs, which affect rpoS expression. It is induced under oxidative stress and reduces the levels of the stationary phase sigma factor RpoS. We analyzed the turn-over of OxyS and rpoS mRNA in early exponential and in stationary growth phase in different E. coli strains to learn more about the mechanisms of processing and about a possible impact of processing on growth-dependent regulation. We could not attribute a major role of RNase E, RNase III, PNPase or RNase II on OxyS turn-over in exponential growth phase. Only the simultaneous lack of RNase E, PNPase and RNase II activity resulted in some stabilization of OxyS in exponential growth phase, implying the action of multiple ribonucleases on OxyS turn-over. A major role of RNase E on OxyS stability was observed in stationary phase and was dependent on the presence of the RNA binding protein Hfq and of DsrA, one of the other small RNAs binding to rpoS mRNA. Our data also confirm a role of RNase III in rpoS turn-over, however, only in exponential growth phase.We conclude that OxyS and rpoS mRNA processing is influenced by different RNases and additional factors like Hfq and DsrA and that the impact of these factors is strongly dependent on growth phase.
 ...
PMID:The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its target rpoS in E. coli is growth phase dependent. 2001 54

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.
 ...
PMID:(p)ppGpp inhibits polynucleotide phosphorylase from streptomyces but not from Escherichia coli and increases the stability of bulk mRNA in Streptomyces coelicolor. 2058 Dec 11

The degradosome is a multienzyme complex involved in mRNA degradation in Escherichia coli. The essential endoribonuclease RNase E contains a large noncatalytic region necessary for protein-protein interactions with other components of the RNA degradosome. Interacting proteins include the DEAD-box RNA helicase RhlB, the glycolytic enzyme enolase, and the exoribonuclease PNPase. Pseudoalteromonas haloplanktis, a psychrotolerant gammaproteobacterium distantly related to E. coli, encodes homologs of each component of the RNA degradosome. In P. haloplanktis, RNase E associates with RhlB and PNPase but not enolase. Plasmids expressing P. haloplanktis RNase E (Ph-RNase E) can complement E. coli strains lacking E. coli RNase E (Ec-RNase E). Ph-RNase E, however, does not confer a growth advantage to E. coli at low temperature. Ph-RNase E has a heterologous protein-protein interaction with Ec-RhlB but not with Ec-enolase or Ec-PNPase. The Ph-RNase E binding sites for RhlB and PNPase were mapped by deletion analysis. The PNPase binding site is located at the C-terminal end of Ph-RNase E at the same position as that in Ec-RNase E, but the sequence of the site is not conserved. The sequence of the RhlB binding site in Ph-RNase E is related to the sequence in Ec-RNase E. Together with the heterologous interaction between Ph-RNase E and Ec-RhlB, our results suggest that the underlying structural motif for the RNase E-RhlB interaction is conserved. Since the activity of Ec-RhlB requires its physical interaction with Ec-RNase E, conservation of the underlying structural motif over a large evolutionary distance could be due to constraints involved in the control of RhlB activity.
 ...
PMID:Characterization of the RNA degradosome of Pseudoalteromonas haloplanktis: conservation of the RNase E-RhlB interaction in the gammaproteobacteria. 2072 66


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>