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 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 More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus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.
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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.
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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.
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PMID:A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli. 1916 40

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

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.
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PMID:Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners. 1934 89

Efficient turnover of unnecessary and misfolded RNAs is critical for maintaining the integrity and function of the mitochondria. The mitochondrial RNA degradosome of budding yeast (mtEXO) has been recently studied and characterized; yet no RNA degradation machinery has been identified in the mammalian mitochondria. In this communication, we demonstrated that purified human SUV3 (suppressor of Var1 3) dimer and polynucleotide phosphorylase (PNPase) trimer form a 330-kDa heteropentamer that is capable of efficiently degrading double-stranded RNA (dsRNA) substrates in the presence of ATP, a task the individual components cannot perform separately. The configuration of this complex is similar to that of the core complex of the E. coli RNA degradosome lacking RNase E but very different from that of the yeast mtEXO. The hSUV3-hPNPase complex prefers substrates containing a 3' overhang and degrades the RNA in a 3'-to-5' directionality. Deleting a short stretch of amino acids (positions 510-514) compromises the ability of hSUV3 to form a stable complex with hPNPase to degrade dsRNA substrates but does not affect its helicase activity. Furthermore, two additional hSUV3 mutants with abolished helicase activity because of disrupted ATPase or RNA binding activities were able to bind hPNPase. However, the resulting complexes failed to degrade dsRNA, suggesting that an intact helicase activity is essential for the complex to serve as an effective RNA degradosome. Taken together, these results strongly suggest that the complex of hSUV3-hPNPase is an integral entity for efficient degradation of structured RNA and may be the long sought RNA-degrading complex in the mammalian mitochondria.
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PMID:Human mitochondrial SUV3 and polynucleotide phosphorylase form a 330-kDa heteropentamer to cooperatively degrade double-stranded RNA with a 3'-to-5' directionality. 1950 88

Here we report a unique processing pathway in Escherichia coli for tRNA(Leu5) in which the exoribonuclease polynucleotide phosphorylase (PNPase) removes the Rho-independent transcription terminator from the leuX transcript without requiring the RhlB RNA helicase. Our data demonstrate for the first time that PNPase can efficiently degrade an RNA substrate containing secondary structures in vivo. Furthermore, RNase P, an endoribonuclease that normally generates the mature 5'-ends of tRNAs, removes the leuX terminator inefficiently independent of PNPase activity. RNase P cleaves 4-7 nt downstream of the CCA determinant generating a substrate for RNase II, which removes an additional 3-4 nt. Subsequently, RNase T completes the 3' maturation process by removing the remaining 1-3 nt downstream of the CCA determinant. RNase E, G and Z are not involved in terminator removal. These results provide further evidence that the E. coli tRNA processing machinery is far more diverse than previously envisioned.
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PMID:Processing of the Escherichia coli leuX tRNA transcript, encoding tRNA(Leu5), requires either the 3'-->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator. 1990 95

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.
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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

CspA, a small protein that is highly induced by cold shock, is encoded by a monocistronic mRNA of 428 nucleotides (nt) whose half-life and abundance are greatly increased following cold shock. We show here that in vitro cspA mRNA can bind multiple copies of Hfq, a hexameric Sm-like protein which promotes a variety of RNA-RNA interactions. Binding of the first Hfq hexamer occurs with an apparent K(d) (dissociation constant) of <40 nM; up to seven additional hexamers can bind sequentially at higher concentrations. Known ligands of Hfq, including the small regulatory RNA, RyhB, compete with cspA mRNA. Several experiments suggest that the first binding site to be occupied by Hfq is located at or near the 3' end of cspA mRNA. The consequences of limited Hfq binding in vitro include nearly total inhibition of RNase E cleavage at a site approximately 35 nt from the 3' end of the mRNA, stimulation of polyadenylation by poly(A) polymerase 1, and subsequent exonucleolytic degradation by polynucleotide phosphorylase. We propose that Hfq may play a facilitating role in the metabolism of cspA mRNA.
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PMID:Interactions of the RNA-binding protein Hfq with cspA mRNA, encoding the major cold shock protein. 2023 32


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