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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 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

The 3'-->5' exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3' extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.
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PMID:Action of RNase II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure. 1076 41

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.
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PMID:Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. 1079 69

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

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.
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PMID:RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli. 1094 97

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

8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.
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PMID:Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein. 1150 94

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

A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease. YhaM protein has a C-terminal "HD domain," found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal "OB-fold," present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.
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PMID:Bacillus subtilis YhaM, a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria. 1239 95

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.
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PMID:RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli. 1260 Oct


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