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:3.1.13.1 (exoribonuclease)
732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. Both enzymes are important in RNA processing and messenger RNA degradation. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.
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PMID:A DEAD-box RNA helicase in the Escherichia coli RNA degradosome. 861 17

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.
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PMID:Proteins associated with RNase E in a multicomponent ribonucleolytic complex. 863 81

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

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.
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PMID:The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly. 975 18

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.
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PMID:Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome. 1168 Aug 51

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. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.
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PMID:Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach. 1613 13

In yeast mitochondria, RNA degradation takes place through the coordinated activities of ySuv3 helicase and yDss1 exoribonuclease (mtEXO), whereas in bacteria, RNA is degraded via RNaseE, RhlB, PNPase, and enolase. Yeast lacking the Suv3 component of the mtEXO form petits and undergo a toxic accumulation of omega intron RNAs. Mammalian mitochondria resemble their prokaryotic origins by harboring a polyadenylation-dependent RNA degradation mechanism, but whether SUV3 participates in regulating RNA turnover in mammalian mitochondria is unclear. We found that lack of hSUV3 in mammalian cells subsequently yielded an accumulation of shortened polyadenylated mtRNA species and impaired mitochondrial protein synthesis. This suggests that SUV3 may serve in part as a component of an RNA degradosome, resembling its yeast ancestor. Reduction in the expression levels of oxidative phosphorylation components correlated with an increase in reactive oxygen species generation, whereas membrane potential and ATP production were decreased. These cumulative defects led to pleiotropic effects in mitochondria such as decreased mtDNA copy number and a shift in mitochondrial morphology from tubular to granular, which eventually manifests in cellular senescence or cell death. Thus, our results suggest that SUV3 is essential for maintaining proper mitochondrial function, likely through a conserved role in mitochondrial RNA regulation.
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PMID:Role of SUV3 helicase in maintaining mitochondrial homeostasis in human cells. 1867 73

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.
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PMID:A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli. 1916 40

Endoribonuclease E, 3'-5' exoribonuclease polynucleotide phosphorylase, RhlB RNA helicase and enolase form a multienzyme complex (the "degradosome") playing an important role in RNA processing and decay in Escherichia coli. Although a number of proteins that occasionally co-purify with the E. coli degradosome in non-stoichiometric amounts were initially viewed as insignificant constituents of this complex, recent studies suggest that, in fact, some of these copurifying proteins are involved in modulation of degradosome composition, activity or specificity during bacterial adaptation to changing environments. Here we briefly review these findings and discuss their implications for understanding the multifaceted mechanisms controlling degradosome functions in vivo.
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PMID:Unraveling new roles for minor components of the E. coli RNA degradosome. 1966 55

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.
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PMID:The critical role of RNA processing and degradation in the control of gene expression. 2067 45


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