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)

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.
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PMID:Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP. 917 91

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.
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PMID:Polyphosphate kinase is a component of the Escherichia coli RNA degradosome. 938 62

We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis. Originally designated mrsC (mRNA stability), the mrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F. Wang et al., J. Bacteriol. 180:1929-1938, 1998). Strains carrying the thermosensitive mrsC505 allele stopped growing soon after the temperature was shifted to 44 degrees C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44 degrees C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant. In an rne-1 mrsC505 double mutant, the average half-life was 19.8 min. Inactivating mrsC significantly increased the half-lives of the trxA, cat, secG, and kan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of mrsC505 multiple mutants at 44 degrees C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.
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PMID:The Escherichia coli mrsC gene is required for cell growth and mRNA decay. 953 93

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

We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings. The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts. More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties. These activities are characteristic of PNPase. Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity. We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study. We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts. The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).
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PMID:Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase. 965 46

ermC mRNA decay was examined in a mutant of Bacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase). 5'-proximal RNA fragments less than 400 nucleotides in length were abundant in the pnpA strain but barely detectable in the wild type. On the other hand, the patterns of 3'-proximal RNA fragments were similar in the wild-type and pnpA strains. Northern blot analysis with different probes showed that the 5' end of the decay intermediates was the native ermC 5' end. For one prominent ermC RNA fragment, in particular, it was shown that formation of its 3' end was directly related to the presence of a stalled ribosome. 5'-proximal decay intermediates were also detected for transcripts encoded by the yybF gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.
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PMID:Decay of ermC mRNA in a polynucleotide phosphorylase mutant of Bacillus subtilis. 981 56

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3' single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.
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PMID:Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase. 1052 3

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

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

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock-induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures. We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10-fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift-like pattern in a PNPase-independent manner. The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs. The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase. In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho-dependent termination site within pnp. In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.
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PMID:Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli. 1093 Dec 96


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