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)

The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.
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PMID:Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli. 1552 87

The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.
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PMID:The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE. 1555 79

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.
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PMID:Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W. 1570 81

The RNA degradosome of Escherichia coli is a ribonucleolytic multienzyme complex containing RNase E, polynucleotide phosphorylase, RhlB, and enolase. Previous in vitro and in vivo work has shown that RhlB facilitates the exonucleolytic degradation of structured mRNA decay intermediates by polynucleotide phosphorylase in an ATPase-dependent reaction. Here, we show that deleting the gene encoding RhlB stabilizes a lacZ mRNA transcribed by bacteriophage T7 RNA polymerase. Deleting the gene encoding enolase has little if any effect. Other messages transcribed by T7 polymerase are also stabilized by DeltarhlB. The effect of point mutations inactivating RhlB is comparable with the effect of deleting the gene. Primer extension analysis of the lacZ message indicates that RhlB facilitates endoribonucleolytic cleavage by RNase E, demonstrating a functional interaction between the RNA helicase and the endoribonuclease. The possible physiological role of an RhlB-RNase E pathway and the mechanisms by which RhlB could facilitate RNase E cleavage are discussed.
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PMID:Evidence in vivo that the DEAD-box RNA helicase RhlB facilitates the degradation of ribosome-free mRNA by RNase E. 1586 49

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

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.
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PMID:Function of the conserved S1 and KH domains in polynucleotide phosphorylase. 1623 5

Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal "scaffold" region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex (alpha(3)beta(2)) containing trimers of a catalytic subunit (alpha) and dimers of another subunit (beta). The beta-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-alpha and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-alpha, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-alpha only with RhlB. Using affinity chromatography, we found that PNPase-alpha and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for alpha(3)beta(2) (i.e., 377-380 kDa), whereas no association between PNPase-alpha and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single- or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-alpha and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-alpha is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the beta-subunit of E. coli PNPase complex.
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PMID:RhlB helicase rather than enolase is the beta-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)-exoribonucleolytic complex. 1627 23

In the present work we have used a double-hybrid assay in bacteria to identify a putative domain in E. coli PNPase required for in vivo interaction with RNase E. We used a 202 aa fragment of RNase E previously reported as the PNPase binding domain in this enzyme and a collection of 13 different fragments of 105 aa, spanning the entire sequence of 734 aa PNPase (GenBank Accession number NP_417633). Our results indicate that two clones of PNPase including residues 158-262 and residues 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration. Three-dimensional modeling of the E. coli PNPase based on the S. antibioticus protein structure indicates that the putative binding domain is located on the monomer surface, facing outward from the trimeric tertiary structure. Since a copy of the betabetaalphabetabetaalpha domain is also found in RNase PH, we investigated and found an interaction with RNase E in a pull-down assay. We suggest this interaction takes place through the similar betabetaalphabetabetaalpha domain present in the tertiary structure of this enzyme. Based on these results, we propose that RNase PH and RNase E could form functional assemblies in E. coli.
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PMID:Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain. 1648 7

In nature, bacteria remain mostly in the stationary phase of the life cycle. Although mRNA is a major determinant of gene expression, little is known about mRNA decay in the stationary phase. The results presented herein demonstrate that RNase R is induced in stationary phase and is involved in the post-transcriptional regulation of ompA mRNA. This work is the first report of RNase R activity on a full length mRNA. In the absence of RNase R in a single rnr mutant, higher levels of ompA mRNA are found as a consequence of the stabilization of ompA full transcript. This effect is growth-phase-specific and not a growth-rate-dependent event. These higher levels of ompA mRNA were correlated with increases in the amounts of OmpA protein. We have also analysed the role of other factors that could affect ompA mRNA stability in stationary phase. RNase E was found to have the most important role, followed by polyadenylation. PNPase also affected the decay of the ompA transcript but RNase II did not seem to contribute much to this degradation process. The participation of RNase R in poly(A)-dependent pathways of decay in stationary phase of growth is discussed. The results show that RNase R can be a modulator of gene expression in stationary phase cells.
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PMID:RNase R affects gene expression in stationary phase: regulation of ompA. 1655 33

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.
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PMID:Exoribonuclease R in Pseudomonas syringae is essential for growth at low temperature and plays a novel role in the 3' end processing of 16 and 5 S ribosomal RNA. 1740 75


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