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)

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 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.
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PMID:The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation. 1465 5

Chloroplasts were acquired by eukaryotic cells through endosymbiosis and have retained their own gene expression machinery. One hallmark of chloroplast gene regulation is the predominance of posttranscriptional control, which is exerted both at the gene-specific and global levels. This review focuses on how chloroplast mRNA stability is regulated, through an examination of poly(A)-dependent and independent pathways. The poly(A)-dependent pathway is catalyzed by polynucleotide phosphorylase (PNPase), which both adds and degrades destabilizing poly(A) tails, whereas RNase II and PNPase may both participate in the poly(A)-independent pathway. Each system is initiated through endonucleolytic cleavages that remove 3' stem-loop structures, which are catalyzed by the related proteins CSP41a and CSP41b and possibly an RNase E-like enzyme. Overall, chloroplasts have retained the prokaryotic endonuclease-exonuclease RNA degradation system despite evolution in the number and character of the enzymes involved. This reflects the presence of the chloroplast within a eukaryotic host and the complex responses that occur to environmental and developmental cues.
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PMID:Cooperation of endo- and exoribonucleases in chloroplast mRNA turnover. 1521 Mar 34

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.
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PMID:Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces. 1531 61

In Escherichia coli, the post-transcriptional addition of poly(A) tails by poly(A) polymerase I (PAP I, pcnB) plays a significant role in cellular RNA metabolism. However, many important features of this system, including its regulation and the selection of polyadenylation sites, are still poorly understood. Here we show that the inactivation of Hfq (hfq), an abundant RNA-binding protein, leads to the reduction in the ability of PAP I to add poly(A) tails at the 3' termini of mRNAs containing Rho-independent transcription terminators even though PAP I protein levels remain unchanged. Those poly(A) tails that are synthesized in the absence of Hfq are shorter in length, even in the absence of polynucleotide phosphorylase (PNPase), RNase II and RNase E. In fact, the biosynthetic activity of PNPase in the hfq single mutant is enhanced and it becomes the primary polynucleotide polymerase, adding heteropolymeric tails almost exclusively to 3' truncated mRNAs. Surprisingly, both PNPase and Hfq co-purified with His-tagged PAP I under native conditions indicating a potential complex among these proteins. Immunoprecipitation experiments using PNPase- and Hfq-specific antibodies confirmed the protein-protein interactions among PAP I, PNPase and Hfq. Analysis of mRNA half-lives in hfq, deltapcnB and hfq deltapcnB mutants suggests that Hfq and PAP I function in the same mRNA decay pathway.
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PMID:The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. 1552 76

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

Maturation of the tRNA 3' terminus is a complicated process in bacteria. Usually, it is initiated by an endonucleolytic cleavage carried out by RNase E and Z in different bacteria. In Escherichia coli, RNase E cleaves AU-rich sequences downstream of tRNA, producing processing intermediates with a few extra residues at the 3' end; these are then removed by exoribonuclease trimming to generate the mature 3' end. Here we show that essentially all E. coli tRNA precursors contain a potential RNase E cleavage site, the AU-rich sequence element (AUE), in the 3' trailer. This suggests that RNase E cleavage and exonucleolytic trimming is a general pathway for tRNA maturation in this organism. Remarkably, the AUE immediately downstream of each tRNA is selectively conserved in bacteria having RNase E and tRNA-specific exoribonucleases, suggesting that this pathway for tRNA processing is also commonly used in these bacteria. Two types of RNase E-like proteins are identified in actinobacteria and the alpha-subdivision of proteobacteria. The tRNA 3' proximal AUE is conserved in bacteria with only one type of E-like protein. Selective conservation of the AUE is usually not observed in bacteria without RNase E. These results demonstrate a novel example of co-evolution of RNA sequences with processing activities.
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PMID:Co-evolution of tRNA 3' trailer sequences with 3' processing enzymes in bacteria. 1581 23

In all living cells 3' ends of RNA are posttranscriptionally elongated or shortened by nucleotidyl transferases and ribonucleases. The detailed analysis of the rpsO mRNA of Escherichia coli presented here demonstrates that transcription terminates in vivo at two sites located seven and eight nucleotides downstream from the GC-rich hairpin of the intrinsic terminator and that primary transcripts can be shortened by RNase II. The shortest RNA identified in the cell result from nibbling of primary transcripts. Primary transcripts and nibbled molecules can also be adenylated by poly(A) polymerase I (PAP I). In addition, kinetics of decay performed in vitro demonstrate that RNase II rapidly degrades poly(A) tails longer than 7-8 As processively while it slowly nibbles shorter tails and non adenylated RNAs distributively. Comparison of the kinetics of nibbling of oligoadenylated rpsO mRNA in vivo and in vitro lead us to conclude that the rates of shortening and elongation of the oligo(A) tails detected in vivo are very slow: about 0.5-7 nucleotides per min. We finally speculate that the slowness of oligo(A) synthesis may explain why polyadenylation does not affect the stability of mRNAs whose degradation is controlled by RNase E.
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PMID:Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease II and poly(A) polymerase. 1588 70

Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.
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PMID:Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels. 1613 83

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


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