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David H Bechhofer - One of the best experts on this subject based on the ideXlab platform.
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properties of a bacillus subtilis Polynucleotide Phosphorylase deletion strain
Journal of Bacteriology, 1996Co-Authors: Wei Wang, David H BechhoferAbstract:The pnpA gene of Bacillus subtilis, which codes for Polynucleotide Phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants. Growth defects of both B. subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene. RNA decay characteristics of the B. subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA. The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, Pi-dependent RNA decay activity of PNPase. In vitro experiments suggested that a previously identified, Mn2+ -dependent hydrolytic activity was important for decay in the pnpA mutant. In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug. The pnpA deletion strain also exhibited multiseptate, filamentous growth. It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes.
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in vitro processing activity of bacillus subtilis Polynucleotide Phosphorylase
Molecular Microbiology, 1996Co-Authors: Sutapa Mitra, Kim K Hue, David H BechhoferAbstract:A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al., 1996 — accompanying paper), this exonuclease activity was shown to be due to Polynucleotide Phosphorylase (PNPase). Processive 3′-to-5′ exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3′ end differed for the B. subtilis and Escherichia coli PNPase enzymes.
Mikael Rhen - One of the best experts on this subject based on the ideXlab platform.
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Opposing Contributions of Polynucleotide Phosphorylase and the Membrane Protein NlpI to Biofilm Formation by Salmonella enterica Serovar Typhimurium
Journal of bacteriology, 2010Co-Authors: Syed Fazle Rouf, Irfan Ahmad, Naeem Anwar, Suman K. Vodnala, Abdul Kader, Ute Römling, Mikael RhenAbstract:CsgD and cyclic-3',5'-di-guanylate are key regulators of biofilm formation in Salmonella enterica serovar Typhimurium. Our results show that Polynucleotide Phosphorylase and NlpI oppositely altered expression of CsgD. Polynucleotide Phosphorylase and NlpI also had opposite effects on the expression of yjcC, which codes for a cyclic-3',5'-di-guanylate phosphodiesterase affecting CsgD expression.
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Polynucleotide Phosphorylase is a global regulator of virulence and persistency in salmonella enterica
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Mark Clements, Sofia Eriksson, Arthur Thompson, Sacha Lucchini, Jay C D Hinton, Staffan Normark, Mikael RhenAbstract: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.
Gianni Dehò - One of the best experts on this subject based on the ideXlab platform.
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regulation of escherichia coli Polynucleotide Phosphorylase by atp
Journal of Biological Chemistry, 2008Co-Authors: Marta Del Favero, Paolo Tortora, Elisa Mazzantini, Federica Briani, Sandro Zangrossi, Gianni DehòAbstract:Polynucleotide Phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.
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genetic analysis of Polynucleotide Phosphorylase structure and functions
Biochimie, 2007Co-Authors: Federica Briani, Paolo Tortora, Marta Del Favero, Sandro Zangrossi, Rossana Capizzuto, Chiara Consonni, Claudio Greco, Luca De Gioia, Gianni DehòAbstract:Polynucleotide Phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.
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Polynucleotide Phosphorylase-based photometric assay for inorganic phosphate.
Analytical biochemistry, 2004Co-Authors: Andrea Ghetta, Maura Matus-ortega, Jaime García-mena, Gianni Dehò, Paolo Tortora, Maria Elena RegonesiAbstract:Polynucleotide Phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of Polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5 0 -diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5 0 -triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaVected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in diVerent biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged Polynucleotide Phosphorylase developed in our laboratories allowed quick and straightforward puriWcation of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.
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Polynucleotide Phosphorylase-Deficient Mutants of Pseudomonas putida
Journal of bacteriology, 2003Co-Authors: Rebecca Favaro, Gianni DehòAbstract:In bacteria, Polynucleotide Phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved. In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature. We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity. In addition, we found that the transcription pattern of pnp upon cold shock in P. putida was markedly different from that in Escherichia coli. It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species.
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photometric assay for Polynucleotide Phosphorylase
Analytical Biochemistry, 1999Co-Authors: Laura Fontanella, Gianni Dehò, Rebecca Favaro, Sabrina Pozzuolo, Alessandra Costanzo, Paolo TortoraAbstract:Polynucleotide Phosphorylase (PNPase) is a prokaryotic enzyme that catalyzes phosphorolysis of Polynucleotides with release of NDPs. It is also believed to play a key role in turnover of prokaryotic transcripts, thus regulating gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [32P]phosphate and poly(A) in the presence of the enzyme, separating [32P]phosphate from [32P]ADP, and quantifying ADP by scintillation counting. Photometric assay using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes is not feasible in crude extracts because of endogenous ATPase activities, which regenerate ADP from the ATP released by pyruvate kinase. Here, we present a simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate. Glucose 6-phosphate is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based on the theoretical model, a linear increase in absorbance is predicted as a function of the square of the reaction time, with a slope proportional to PNPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We also devised a simple procedure for determining absolute enzyme activities (expressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards.
George H. Jones - One of the best experts on this subject based on the ideXlab platform.
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Novel Aspects of Polynucleotide Phosphorylase Function in Streptomyces.
Antibiotics (Basel Switzerland), 2018Co-Authors: George H. JonesAbstract:Polynucleotide Phosphorylase (PNPase) is a 3′–5′-exoribnuclease that is found in most bacteria and in some eukaryotic organelles. The enzyme plays a key role in RNA decay in these systems. PNPase structure and function have been studied extensively in Escherichia coli, but there are several important aspects of PNPase function in Streptomyces that differ from what is observed in E. coli and other bacterial genera. This review highlights several of those differences: (1) the organization and expression of the PNPase gene in Streptomyces; (2) the possible function of PNPase as an RNA 3′-polyribonucleotide polymerase in Streptomyces; (3) the function of PNPase as both an exoribonuclease and as an RNA 3′-polyribonucleotide polymerase in Streptomyces; (4) the function of (p)ppGpp as a PNPase effector in Streptomyces. The review concludes with a consideration of a number of unanswered questions regarding the function of Streptomyces PNPase, which can be examined experimentally.
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Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a Polynucleotide Phosphorylase
Journal of bacteriology, 1996Co-Authors: George H. Jones, M J BibbAbstract:The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to Polynucleotide Phosphorylase from Escherichia coli. Indeed, like E. coli Polynucleotide Phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, the E. coli enzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the cloned gpsI gene in E. coli led to an increase in both Polynucleotide Phosphorylase and guanosine pentaphosphate synthetase activities in the cloning host. The Polynucleotide Phosphorylase activities of GPSI and of the E. coli enzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and Polynucleotide Phosphorylase activities.
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Guanosine Pentaphosphate Synthetase fromStreptomyces antibioticusIs Also a Polynucleotide Phosphorylase
1996Co-Authors: George H. Jones, Andmervyn J. BibbAbstract:The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) fromStreptomyces antibioticushas been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to Polynucleotide Phosphorylase fromEscherichia coli. Indeed, likeE. coliPolynucleotide Phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, theE. colienzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the clonedgpsIgene inE. coli ledtoanincreaseinbothPolynucleotidePhosphorylaseandguanosinepentaphosphatesynthetaseactivitiesin the cloning host. The Polynucleotide Phosphorylase activities of GPSI and of theE. colienzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and Polynucleotide Phosphorylase activities. One of the most important experimental challenges related
Stanley N. Cohen - One of the best experts on this subject based on the ideXlab platform.
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The Streptomyces coelicolor Polynucleotide Phosphorylase Homologue, and Not the Putative Poly(A) Polymerase, Can Polyadenylate RNA
Journal of Bacteriology, 2003Co-Authors: Björn Sohlberg, Jianqiang Huang, Stanley N. CohenAbstract:A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40: 1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor Polynucleotide Phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.