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 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

The stability of mRNA in prokaryotes depends on multiple factors and it has not yet been possible to describe the process of mRNA degradation in terms of a unique pathway. However, important advances have been made in the past 10 years with the characterization of the cis-acting RNA elements and the trans-acting cellular proteins that control mRNA decay. The trans-acting proteins are mainly four nucleases, two endo- (RNase E and RNase III) and two exonucleases (PNPase and RNase II), and poly(A) polymerase. RNase E and PNPase are found in a multienzyme complex called the degradosome. In addition to the host nucleases, phage T4 encodes a specific endonuclease called RegB. The cis-acting elements that protect mRNA from degradation are stable stem-loops at the 5' end of the transcript and terminators or REP sequences at their 3' end. The rate-limiting step in mRNA decay is usually an initial endonucleolytic cleavage that often occurs at the 5' extremity. This initial step is followed by directional 3' to 5' degradation by the two exonucleases. Several examples, reviewed here, indicate that mRNA degradation is an important step at which gene expression can be controlled. This regulation can be either global, as in the case of growth rate-dependent control, or specific, in response to changes in the environmental conditions.
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PMID:Messenger RNA stability and its role in control of gene expression in bacteria and phages. 1069 Apr 8

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single-stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3' termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.
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PMID:Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. 1084 84

RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.
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PMID:RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease E. 1113 27

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.
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PMID:Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli. 1132 69

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.
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PMID:Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E. 1139 Mar 93

Expression of thrS, the gene encoding Escherichia coli threonyl-tRNA synthetase, is negatively autoregulated at the translational level. Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site called the operator, located immediately upstream of the initiation codon. The present work investigates the relationship between regulation and mRNA degradation. We show that two regulatory mutations, which increase thrS expression, cause an increase in the steady-state mRNA concentration. Unexpectedly, however, the half-life of thrS mRNA in the derepressed mutants is equal to that of the wild-type, indicating that mRNA stability is independent of the repression level. All our results can be explained if one assumes that thrS mRNA is either fully translated or immediately degraded. The immediately degraded RNAs are never detected due to their extremely short half-lives, while the fully translated messengers share the same half-lives, irrespective of the mutations. The increase in the steady-state level of thrS mRNA in the derepressed mutants is simply explained by an increase in the population of translated molecules, i.e. those never bound by the repressor, ThrRS. Despite this peculiarity, thrS mRNA degradation seems to follow the classical degradation pathway. Its stability is increased in a strain defective for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation. We also observe an accumulation of small fragments corresponding to the 5' end of the message in a strain defective for polynucleotide phosphorylase, indicating that, following the endonucleolytic cleavages, fragments are normally degraded by 3' to 5' exonucleolytic trimming. Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS.
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PMID:The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene. 1145 82

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

mRNA instability is an intrinsic property that permits timely changes in gene expression by limiting the lifetime of a transcript. The RNase e of Escherichia coli is a single-strand-specific endo-nuclease involved in the processing of rRNA and the degradation of mRNA. A nucleolytic multi-enzyme complex now known as the RNA degradosome was discovered during the purification and characterization of RNase E. Two other components are a 3' exoribonuclease (polynucleotide phosphorylase, PNPase) and a DEAD-box RNA helicase (RNA helicase B, RhlB). RNase E is a large multidomain protein with N-terminal ribonucleolytic activity, an RNA-binding domain and a C-terminal "scaffold" that binds PNPase, enolase and RhlB. RhlB by itself has little activity but is strongly stimiulated by its interaction with RNase E. RhlB in vitro can facilitate the degradation of structured RNA by PNPase. Since the discovery of the RNA degradosome in E. coli, related complexes have been described in other organisms.
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PMID:The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes. 1203 60

Bacteriophage P4 immunity is controlled by a small stable RNA (CI RNA) that derives from the processing of primary transcripts. In previous works, we observed that the endonuclease RNase P is required for the maturation of CI RNA 5'-end; moreover, we found that polynucleotide phosphorylase (PNPase), a 3' to 5' RNA-degrading enzyme, is required for efficient 5'-end processing of CI RNA, suggesting that 3'-end degradation of the primary transcript might be involved in the production of proper RNase P substrates. Here, we demonstrate that another Escherichia coli nuclease, RNase E, would appear to be involved in this process. We found that transcripts of the P4 immunity region are modified by the post-transcriptional addition of short poly(A) tails and heteropolymeric tails with prevalence of A residues. Most oligoadenylated transcripts encompass the whole cI locus and are thus compatible as intermediates in the CI RNA maturation pathway. On the contrary, in a polynucleotide phosphorylase (PNPase)-defective host, adenylation occurred most frequently within cI, implying that such transcripts are targeted for degradation. We did not find polyadenylation in a pcnB mutant, suggesting that the pcnB-encoded polyadenyl polymerase I (PAP I) is the only enzyme responsible for modification of P4 immunity transcripts. Maturation of CI RNA 5'-end in such a mutant was impaired, further supporting the idea that processing of the 3'-end of primary transcripts is an important step for efficient maturation of CI RNA by RNase P.
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PMID:RNase E and polyadenyl polymerase I are involved in maturation of CI RNA, the P4 phage immunity factor. 1205 40


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