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At least 667 records · Page 37Linked to original sources
PubMed1999

A novel mutation in the KH domain of polynucleotide phosphorylase affects autoregulation and mRNA decay in Escherichia coli.

Polynucleotide phosphorylase (PNPase) is a key 3'-5' exonuclease for mRNA decay in bacteria. Here, we report the isolation of a novel mutant of Escherichia coli PNPase that affects autogenous control and mRNA decay. We show that the inactivation of PNPase by a transposon insertion increases the half-life of galactokinase mRNA encoded by a plasmid. When the bacteriophage lambda int gene retroregulator (sib/tI ) is placed between pgal and galK, it severely diminishes galactokinase expression because of transcription termination. The expression of galK from this construct is increased by a single base mutation, sib1, which causes a partial readthrough of transcription at tI. We have used this plasmid system with sib1 to select E. coli mutants that depress galK expression. Genetic and molecular analysis of one such mutant revealed that it contains a mutation in the pnp gene, which encodes the PNPase catalytic subunit alpha. The mutation responsible (pnp-71 ) has substituted a highly conserved glycine residue in the KH domain of PNPase with aspartate. We show that this G-570D substitution causes a higher accumulation of the alpha-subunit as a result of defective autoregulation, thereby increasing the PNPase activity in the cell. The purified mutant alpha-subunit shows the same electrophoretic mobility in denaturing gels as the wild-type subunit, as expected. However, the mutant protein present in crude extracts displays an altered electrophoretic mobility in non-denaturing gels that is indicative of a novel enzyme complex. We present a model for how the pnp-71 mutation might affect autoregulation and mRNA decay based on the postulated role of the KH domain in RNA-protein and protein-protein interactions.

Amino Acid Sequence↗
PubMed2000

Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli.

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.

Endoribonucleases↗
PubMed2000

Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli.

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.

Acclimatization↗
PubMed2001

Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation.

When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.

Adaptation, Biological↗
PubMed2002

Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E.

Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half-lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp-lacZ and rne-lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5' untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.

5' Untranslated Regions↗
PubMed2003

Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay.

Previous work has shown that simultaneous inactivation of polynucleotide phosphorylase (PNPase) and RNase II (both 3' 5' exonucleases) in Escherichia coli leads to the loss of cell viability and the accumulation of partially degraded mRNA species. In order to help to distinguish how these two enzymes globally affect the abundance and decay of mRNAs, we have carried out a genome-wide analysis of the steady-state levels of E. coli transcripts using deletion mutations in either rnb or pnp. The data show that, in exponentially growing cells, inactivation of PNPase leads to an increase in the steady-state level of more expressed mRNAs (17.3%) than inactivation of RNase II (7.3%). In contrast, the steady-state levels of a large number of E. coli mRNAs (31%) are decreased in the absence of RNase II, including almost all the ribosomal protein genes, suggesting that a major function of this enzyme is to protect specific mRNAs from the activity of other ribonucleases. Array data were confirmed by Northern analysis of 12 individual mRNAs. A comparison between the steady-state levels and the half-lives of individual mRNAs indicates that there may be a direct interaction between transcription and mRNA decay for some of the transcripts. In addition, results are presented to show significant phenotypic differences between the pnp-7 point mutant and the pnp delta 683 deletion allele.

Blotting, Northern↗
PubMed1999

Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.

Escherichia coli↗
PubMed2005

RhlB helicase rather than enolase is the beta-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)-exoribonucleolytic complex.

Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal "scaffold" region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex (alpha(3)beta(2)) containing trimers of a catalytic subunit (alpha) and dimers of another subunit (beta). The beta-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-alpha and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-alpha, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-alpha only with RhlB. Using affinity chromatography, we found that PNPase-alpha and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for alpha(3)beta(2) (i.e., 377-380 kDa), whereas no association between PNPase-alpha and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single- or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-alpha and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-alpha is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the beta-subunit of E. coli PNPase complex.

Base Sequence↗
PubMed2003

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R.

Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42 degrees C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31 degrees C or when single mutant strains were grown at 42 degrees C. Pulse-chase analysis showed that the rRNA fragments appeared within 5 min at 42 degrees C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.

Base Sequence↗
PubMed2002

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica.

For many pathogens, the ability to regulate their replication in host cells is a key element in establishing persistency. Here, we identified a single point mutation in the gene for polynucleotide phosphorylase (PNPase) as a factor affecting bacterial invasion and intracellular replication, and which determines the alternation between acute or persistent infection in a mouse model for Salmonella enterica infection. In parallel, with microarray analysis, PNPase was found to affect the mRNA levels of a subset of virulence genes, in particular those contained in Salmonella pathogenicity islands 1 and 2. The results demonstrate a connection between PNPase and Salmonella virulence and show that alterations in PNPase activity could represent a strategy for the establishment of persistency.

Animals↗
PubMed2002

Identification and cloning of human polynucleotide phosphorylase, hPNPase old-35, in the context of terminal differentiation and cellular senescence.

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPase(old-35)), that is regulated predominantly by type I IFNs. The hPNPase(OLD-35) protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPase(old-35) in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPase(old-35), an IFN-inducible 3'-5' RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

Amino Acid Sequence↗
PubMed1971

A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma 180 ascites cells.

The rapidly-labeled polyribosomal RNA component from mouse sarcoma 180 cells is retained on nitrocellulose (Millipore) membrane-filters at high ionic strength. This property is due to the presence of a polynucleotide sequence rich in adenylic acid that resists both T(1) and pancreatic RNase digestion. The resistant material shows sedimentation characteristics close to those of transfer RNA. The RNA molecules that contain this material can be separated from the rest of the polysomal RNA by differential phenol extraction with neutral and alkaline Tris buffers. Synthetic poly(A) exhibits the same behavior as the rapidly-labeled polysomal RNA with respect to Millipore binding and phenol fractionation. The characteristics of the rapidly-labeled polysomal RNA component permit its isolation free of ribosomal RNA.

Adenine Nucleotides↗
PubMed1976

A possible conformation for double-stranded polynucleotides.

A model is presented for double-stranded polynucleotides which involves side-by-side meshing of the two strands rather than double helical intertwining. The sugar-phosphate backbone has a twisted strip-like character, yet base-pairing of the Watson-Crick type is still possible. Structural features of the basic model are described and coordinates are presented for a representative example. The structure has, on the whole, reasonable sterochemical contacts, and can be shown to produce a fiber diffraction pattern with x-rays not unlike that of the B form of DNA.

DNA↗
PubMed1978

Preparation and template activities of polynucleotides containing O2- and O4-alkyluridine.

O2-Ethyl-UDP and O4-methyl-UDP have been prepared and copolymerized in various proportions with UDP or CDP, using polynucleotide phosphorylase. The copolymers were used as templates for DNA-dependent RNA polymerases in the presence of Mn2+. Both of the O-alkylated uridines caused a similar misincorporations. When copolymerized with U they led to incorporation of CMP and GMP into the poly(A). No AMP or UMP incorporation seemed to be caused by the introduction of O-alkyluridines into either poly(U) or poly(C). The mispairing of O2- and O4-alkyluridine to behave like C or G represents mutagenic events. O2 alkylation of U or T is, in contrast to O4 alkylation, a relatively frequent result of treatment of double-stranded nucleic acids with N-nitroso alkylating agents. In single-stranded nucleic acids both O2 and O4 alkylations of U and T occur to similar extents. Thus, the observed mutagenic effects of O2 and O4 alkylation of U may be involved in the high carcinogenicity of these alkylating agents.

Alkylation↗
PubMed1980

Nature of the open state in long polynucleotide double helices: possibility of soliton excitations.

The existence of transiently open states in DNA and synthetic polynucleotide double helices has been demonstrated by hydrogen exchange measurements; base pairs reversibly separate and reclose, exposing nucleotide protons to exchange with solvent protons. Recently it has been possible to define the equilibrium, kinetic, and activation parameters of the major open state that determines base pair hydrogen exchange. However, there is no direct information at the moment about the conformation of the open form. Here we consider the possibility that the low energy and slow opening and closing rates observed reflect a deformation involving several adjacent base pairs. Assuming a mobile open unit capable of diffusing along the double helix, we find that available data are consistent with structures of 10 or so adjacent open pairs. It is further suggested that these structures correspond to thermally induced soliton excitations of the double helix, which retain coherence by sharing the energy of a twist deformation among several base pairs. Solitons are nonlinear excitations that can travel as coherent solitary waves, and have been recognized as an important mechanism for mediating conformational changes in polymers and condensed systems generally. Comparison of the double helix with simple mechanical analogs suggests that soliton excitations may well exist within DNA chains, and the present analysis shows that the hydrogen exchange open state is consistent with these.

Base Sequence↗
PubMed1986

Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12.

The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.

Drug Stability↗
PubMed1987

Formation of the triple-stranded polynucleotide helix, poly(A.A.U).

A polynucleotide helical structure containing two strands of poly(A) and one of poly(U) is reported. As shown by spectroscopic observations, the complex only forms when the poly(A) strands are of Mr between 9000 and 50,000 (degree of polymerization congruent to 28-150), whereas the size of the poly(U) strand has no effect. This limitation may explain why poly(A.A.U) was not seen in previous investigations. The potential of the poly(A) tails of mRNA for formation of this triple helix and of A.A.U or/and A.A.T triplet formation to contribute to the binding of specific RNA strands to gene-encoding nucleic acid double helices are noted.

Circular Dichroism↗
PubMed1988

RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase.

Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism. However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally. Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate. Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH. Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing. Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity. RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates. The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.

Bacterial Proteins↗