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 effect of 3'-exoribonucleases on the polyadenylation of mRNA in Escherichia coli was studied by comparing the synthesis and levels of poly(A) RNA in wild-type E coli and mutant strains defective in the two major 3'-exoribonucleases: polynucleotide phosphorylase and ribonuclease II. Mutations which substantially reduced the activity of these 3'-exonucleases caused a 10-fold increase in pulse-labeling of total poly(A) RNA in intact cells. When the net rate of RNA synthesis was measured in permeabilized cells, the mutant with defective 3'-exonucleases showed 20- to 60-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes in non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable cells under conditions when 3'-exoribonucleases were inactive showed a 6-fold higher rate of poly(A) synthesis in the exonuclease-deficient mutant strain, suggesting a higher concentration of mRNA 3'-ends amenable to polyadenylation. Steady-state levels of poly(A) RNA, measured by the ability to serve as template for oligo(dT)-dependent complementary DNA synthesis, also increased more than 40-fold when the 3'-exonucleases were inactivated. Monitoring of the length of the poly(A) tracts by denaturing polyacrylamide gel electrophoresis showed chain lengths of up to 45 residues in the 3'-exonuclease-deficient mutant, whereas most of the poly(A) tracts in the parent strain were shorter than 12 residues. These results show that 3'-exonucleases reduce the level of polyadenylated mRNA in E coli not merely by causing its degradation but also by reducing its rate of synthesis, presumably by competing with poly(A) polymerase for the 3'-ends of mRNA.
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PMID:Polyadenylated mRNA in Escherichia coli: modulation of poly(A) RNA levels by polynucleotide phosphorylase and ribonuclease II. 924 86

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3'-5' exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.
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PMID:The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R. 960 4

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPgammaS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3'-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3' termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.
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PMID:Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes. 964 84

RNA decay in bacteria is carried out by a number of enzymes that participate in the coordinated degradation of their substrates. Endo- and exonucleolytic cleavages as well as polyadenylation are generally involved in determining the half-life of RNAs. Small, untranslated antisense RNAs are suitable model systems to study decay. A study of the pathway of degradation of CopA, the copy number regulator RNA of plasmid R1, is reported here. Strains carrying mutations in the genes encoding RNase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) polymerase I (PcnB/PAP I)--alone or in combination--were used to investigate degradation patterns and relative half-lives of CopA. The results obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3'-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3'-end from PNPase-dependent degradation. Other RNases are probably involved in decay, since in rnb/pnp double mutants, decay still occurs, albeit at a reduced rate. Experiments using purified RNase E identified cleavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell. A model of CopA decay is presented and discussed.
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PMID:Degradation pathway of CopA, the antisense RNA that controls replication of plasmid R1. 969 24

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.
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PMID:Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome. 973 74

Metabolic instability is a hallmark property of mRNAs in most if not all organisms and plays an essential role in facilitating rapid responses to regulatory cues. This article provides a critical examination of recent progress in the enzymology of mRNA decay in Escherichia coli, focusing on six major enzymes: RNase III, RNase E, polynucleotide phosphorylase, RNase II, poly(A) polymerase(s), and RNA helicase(s). The first major advance in our thinking about mechanisms of RNA decay has been catalyzed by the possibility that mRNA decay is orchestrated by a multicomponent mRNA-protein complex (the "degradosome"). The ramifications of this discovery are discussed and developed into mRNA decay models that integrate the properties of the ribonucleases and their associated proteins, the role of RNA structure in determining the susceptibility of an RNA to decay, and some of the known kinetic features of mRNA decay. These models propose that mRNA decay is a vectorial process initiated primarily at or near the 5' terminus of susceptible mRNAs and propagated by successive endonucleolytic cleavages catalyzed by RNase E in the degradosome. It seems likely that the degradosome can be tethered to its substrate, either physically or kinetically through a preference for monphosphorylated RNAs, accounting for the usual "all or none" nature of mRNA decay. A second recent advance in our thinking about mRNA decay is the rediscovery of polyadenylated mRNA in bacteria. Models are provided to account for the role of polyadenylation in facilitating the 3' exonucleolytic degradation of structured RNAs. Finally, we have reviewed the documented properties of several well-studied paradigms for mRNA decay in E. coli. We interpret the published data in light of our models and the properties of the degradosome. It seems likely that the study of mRNA decay is about to enter a phase in which research will focus on the structural basis for recognition of cleavage sites, on catalytic mechanisms, and on regulation of mRNA decay.
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PMID:Degradation of mRNA in Escherichia coli: an old problem with some new twists. 993 52

The rpsO mRNA of E. coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I. We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E. It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II. A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2. Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells. Poly(A)polymerase I contributes differently to the instability of these fragments. The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript. We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA.
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PMID:E. coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent degradation of the mRNA. 1004 80

mRNA decay in prokaryotic cells involves the action of both endo- and exoribonucleases. In Escherichia coli, degradation of RNA to the mononucleotide level was thought to depend on RNase II and polynucleotide phosphorylase. Here, we show that the enzyme oligoribonuclease is an essential part of this process as well. Thus, inactivation of the orn gene encoding oligoribonuclease leads to a cessation of cell growth. Moreover, although pulse-labeled RNA decays normally in orn mutant cells under nonpermissive conditions, a large fraction of the resulting products is small oligoribonucleotides rather than the mononucleotides generated in wild-type cells. The oligoribonucleotides that accumulate are 2-5 residues in length; longer molecules disappear during the decay process. These data indicate that oligoribonuclease is required to complete the degradation of mRNA to mononucleotides and that this process is required for cell viability. Inasmuch as close homologues of the orn gene are found in a wide range of eukaryotes, extending up to humans, these findings raise the possibility that oligoribonuclease also participates in mRNA degradation in these organisms.
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PMID:Oligoribonuclease is an essential component of the mRNA decay pathway. 1020 Feb 69

RNAI is a short RNA, 108 nt in length, which regulates the replication of the plasmid ColE1. RNAI turns over rapidly, enabling plasmid replication rate to respond quickly to changes in plasmid copy number. Because RNAI is produced in abundance, is easily extracted and turns over quickly, it has been used as a model for mRNA in studying RNA decay pathways. The enzymes polynucleotide phosphorylase, poly(A) polymerase and RNase E have been demonstrated to have roles in both messenger and RNAI decay; it is reported here that these enzymes can work independently of one another to facilitate RNAI decay. The roles in RNAI decay of two further enzymes which facilitate mRNA decay, the exonuclease RNase II and the endonuclease RNase III, are also examined. RNase II does not appear to accelerate RNAI decay but it is found that, in the absence of RNase III, polyadenylated RNAI, unprocessed by RNase E, accumulates. It is also shown that RNase III can cut RNAI near nt 82 or 98 in vitro. An RNAI fragment corresponding to the longer of these can be found in extracts of an mc+ pcnB strain (which produces RNase III) but not of an rnc pcnB strain, suggesting that RNAI may be a substrate for RNase III in vivo. A possible pathway for the early steps in RNAI decay which incorporates this information is suggested.
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PMID:Absence of RNASE III alters the pathway by which RNAI, the antisense inhibitor of ColE1 replication, decays. 1058 16

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
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PMID:Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. 1059 33


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