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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 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 RNA degradosome is a multiprotein complex containing an endoribonuclease, polynucleotide phosphorylase and a DEAD-box RNA helicase. A related complex has been described in the spinach chloroplast. The exosome and the mtEXO complex have recently been described in yeast and it is likely that related complexes also exist in animal cells. This research suggests the widespread existence of sophisticated machines for the efficient degradation of messenger RNA. The DEAD-box helicase in the degradosome can unwind regions of RNA structure that interfere with 3'-5' degradation. The polyadenylation of RNA 3' ends is also known to promote degradation by creating a 'toehold' for the degradation machinery. Much remains to be learned about the regulation of mRNA stability. The complexity of the degradation process, both in the eubacteria and in the eukaryotes, suggests that many steps are possible points of control.
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PMID:mRNA degradation. A tale of poly(A) and multiprotein machines. 1008 30

The amount of a messenger RNA available for protein synthesis depends on the efficiency of its transcription and stability. The mechanisms of degradation that determine the stability of mRNAs in bacteria have been investigated extensively during the last decade and have begun to be better understood. Several endo- and exoribonucleases involved in the mRNA metabolism have been characterized as well as structural features of mRNA which account for its stability have been determined. The most important recent developments have been the discovery that the degradosome-a multiprotein complex containing an endoribonuclease (RNase E), an exoribonuclease (polynucleotide phosphorylase), and a DEAD box helicase (RhlB)-has a central role in mRNA degradation and that oligo(A) tails synthesized by poly(A) polymerase facilitate the degradation of mRNAs and RNA fragments. Moreover, the phosphorylation status and the base pairing of 5' extremities, together with 3' secondary structures of transcriptional terminators, contribute to the stability of primary transcripts. Degradation of mRNAs can follow several independent pathways. Interestingly, poly(A) tails and multienzyme complexes also control the stability and the degradation of eukaryotic mRNAs. These discoveries have led to the development of refined models of mRNA degradation.
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PMID:Degradation of mRNA in bacteria: emergence of ubiquitous features. 1068 83

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 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 DEAD-box RNA helicases are a ubiquitous family of enzymes involved in processes that include RNA splicing, ribosome biogenesis, and mRNA degradation. In general, these enzymes help to unwind short stretches of double-stranded RNA in processes that involve the remodeling of RNA structure or of ribonucleoprotein complexes. Here we describe work from our laboratory on the characterization of the RhlB of Escherichia coli, a DEAD-box RNA helicase that is part of a multienzyme complex known as the RNA degradosome. RhlB interacts physically and functionally with RNase E and polynucleotide phosphorylase (PNPase), two other components of the RNA degradosome. We describe enzyme assays that demonstrated that the interaction between RhlB and RNase E is necessary for the ATPase and RNA unwinding activities of RhlB. We also describe an mRNA degradation assay that showed that RhlB facilitates the degradation of structured mRNA by PNPase. These assays are discussed in the context of how they have contributed to our understanding of the function of RhlB in mRNA degradation.
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PMID:Assaying DEAD-box RNA helicases and their role in mRNA degradation in Escherichia coli. 1916 44

The RNA degradosome is a multienzyme complex that plays a key role in the processing of stable RNAs, the degradation of mRNAs, and the action of small regulatory RNAs. Initially discovered in Escherichia coli, similar or related complexes are found in other bacteria. The core of the RNA degradosome is the essential endoribonuclease, RNase E. The C-terminus of this enzyme serves as a scaffold to which other components of the RNA degradosome bind. These ligands include the phosphorolytic 3'-exonuclease, polynucleotide phosphorylase, the DEAD-box RNA helicase, RhlB, and the glycolytic enzyme, enolase. In addition, the DEAD-box RNA helicases CsdA and RhlE and the RNA binding protein, Hfq, may bind to RNase E in place of one or more of the prototypical components. This chapter describes purification of RNase E (the Rne protein), reconstitution of a minimal degradosome that recapitulates the activity of authentic degradosomes, and methods for the assay of the reconstituted complex.
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PMID:Preparation of the Escherichia coli RNase E protein and reconstitution of the RNA degradosome. 1916 45

In Escherichia coli, the cold shock response occurs when there is a temperature downshift from 37 degrees C to 15 degrees C, and this response is characterized by induction of several cold shock proteins, including the DEAD-box helicase CsdA, during the acclimation phase. CsdA is involved in a variety of cellular processes. Our previous studies showed that the helicase activity of CsdA is critical for its function in cold shock acclimation of cells and that the only proteins that were able to complement its function were another helicase, RhlE, an RNA chaperone, CspA, and a cold-inducible exoribonuclease, RNase R. Interestingly, other major 3'-to-5' processing exoribonucleases of E. coli, such as polynucleotide phosphorylase and RNase II, cannot complement the cold shock function of CsdA. Here we carried out a domain analysis of RNase R and showed that this protein has two distinct activities, RNase and helicase, which are independent of each other and are due to different domains. Mutant RNase R proteins that lack the RNase activity but exhibit the helicase activity were able to complement the cold shock function of CsdA, suggesting that only the helicase activity of RNase R is essential for complementation of the cold shock function of CsdA. We also observed that in vivo deletion of the two cold shock domains resulted in a loss of the ability of RNase R to complement the cold shock function of CsdA. We further demonstrated that RNase R exhibits helicase activity in vitro independent of its RNase activity. Our results shed light on the unique properties of RNase R and how it is distinct from other exoribonucleases in E. coli.
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PMID:Escherichia coli RNase R has dual activities, helicase and RNase. 2002 28

In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein-protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.
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PMID:The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex. 2057 37


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