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Information on EC 2.7.7.8 - polyribonucleotide nucleotidyltransferase and Organism(s) Escherichia coli and UniProt Accession P05055
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2.7.7.8 polyribonucleotide nucleotidyltransferaseIUBMB Comments
ADP, IDP, GDP, UDP and CDP can act as donors.
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Word Map
- 2.7.7.8
- rnase
- exoribonuclease
- polya
- ribonuclease
- polymerization
-
polyadenylation
-
phosphorolysis
-
degradosome
- helicase
-
exonuclease
-
exonucleolytic
-
stem-loop
- luteus
- micrococcus
- 5'-diphosphate
-
rna-binding
- kh
- hfq
- oligoribonucleotides
-
rna-degrading
-
heteropolymeric
- antibioticus
-
dead-box
- lysodeikticus
-
primer-independent
- synthesis
- molecular biology
- medicine
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
polynucleotide phosphorylase, hpnpase(old-35), pnpt1, polyribonucleotide nucleotidyltransferase, atmtpnpase, cppnpase, chloroplast pnpase, hpnpaseold-35, polyribonucleotide phosphorylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
nucleoside diphosphate:polynucleotidyl transferase
-
-
-
-
nucleotidyltransferase, polyribonucleotide
-
-
-
-
PNPase
polynucleotide phosphorylase
polyribonucleotide phosphorylase
-
-
-
-
PNPase
-
-
-
-
polynucleotide phosphorylase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
RNAn+1 + phosphate = RNAn + a nucleoside diphosphate
stem-loops of 7,9 or 11 bp block the processive 3'-5' exonuclease action of PNPase
-
RNAn+1 + phosphate = RNAn + a nucleoside diphosphate
mechanism of elongation, overview
-
RNAn+1 + phosphate = RNAn + a nucleoside diphosphate
PNPase exhibits 3'-to-5' exonucleolytic activity
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
nucleotidyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
polyribonucleotide:phosphate nucleotidyltransferase
ADP, IDP, GDP, UDP and CDP can act as donors.
CAS REGISTRY NUMBER
COMMENTARY
9014-12-4
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
rabbit globin mRNAn+1 + phosphate
ADP + rabbit globin mRNAn
-
only the poly(A) tail of the mRNA is removed
-
?
ribonucleoside 5'-diphosphate + phosphate
ribonucleoside 5'-diphosphate + phosphate
-
-
exchange reaction
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
specificity overview
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
specificity overview
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of IDP
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of CDP
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of GDP
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of ADP
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of ADP
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
polymerization of ADP
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
de novo synthesis of polynucleotides, each of the 4 common ribonucleoside diphosphates can serve separately as a substrate for the polymerization reaction, leading to the formation of homopolymers, polymerization of a mixture of nucleoside diphosphates containing different bases results in the formation of a random copolymer, the enzyme does not require a template and cannot copy one, elongation of a primer oligonucleotide with at least 2 nucleoside residues and a free 3'-terminal hydroxyl group, in the reverse reaction breakdown of polyribonucleotides by phosphorolytic cleavage of the internucleotide bonds
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
either in the form of a homotrimeric enzyme or associated in a multiprotein complex, the degradosome, PNPase is involved in RNA processing
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase and RNAse II play an essential role in degrading fragments of mRNA generated by prior cleavage by endonucleases
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase accounts for 10% of total mRNA decay, PNPase can bind double stranded DNA, however the affinity is lower than that obtained for both RNA and single stranded DNA binding
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase synthesizes long, highly heteropolymeric poly(A) tails in vivo and accounts for all of the residual polyadenylylation in poly(A) polymerase deficient strains, in addition PNPase is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing wild-type cultures
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase exonuclease activity plays an essential role in tRNA, mRNA and ribosome metabolism
-
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase is involved in RNA degradation
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase specifically binds to 8-oxoguanine-containing RNA, it is suggested that PNPase discriminate between oxidized and normal RNA which my contribute to a high fidelity of translation
-
-
?
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
phosphorolysis of poly(I)
-
r
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
PNPase prefers degradation of polyadenylated and polyuridinylated RNAs due to the high binding affinities for poly(A) and poly(U), no activity with polyguanylated RNA
-
?
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
processive phosphorolysis of the poly(A) tail of each globin mRNA chain
-
r
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
phosphorolysis of poly(A)
-
r
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
in addition to its degradative role, PNPase can also function as a polymerase, adding 3' tails to transcripts. The reverse of degradation is favored when nucleoside diphosphate rather than inorganic phosphate is present in excess
-
-
r
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
substrate is synthetic radiolabeled SL9A RNA
-
-
?
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
substrates used for the forward degradation reaction are poly(rA) 15-mer RNA and phosphate
substrates used for the reverse polymerization reaction are poly(rA) 15-mer RNA and ADP
-
r
additional information
?
-
regulates its own expression at the level of mRNA stability and translation
-
-
?
additional information
?
-
-
regulates its own expression at the level of mRNA stability and translation
-
-
?
additional information
?
-
-
suppression of Rho-dependent transcription termination within the enzyme gene and its restoration by enzyme protein is an autogenous regulation circuit that modulates enzyme gene expression during cold acclimation
-
-
?
additional information
?
-
-
polynucleotide phosphorylase is essential for growth at low temperatures, while polymerization activity is not essential. RNase PH domains 1 and 2 of polynucleotide phosphorylase are important for its cold shock function, suggesting that the RNase activity of the enzyme is critical for its essential function at low temperature. Its polymerization activity is dispensable in its cold shock function. The RNase R , which is cold inducible, cannot complement the cold shock function of PNPase
-
-
?
additional information
?
-
-
polyribonucleotide phosphorylase-mediated degradation is a major regulatory event controlling the levels of sRNAs, namely stationary phase regulators MicA and RybB, that are required for the accurate expression of outer membrane proteins. Degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. Polyribonucleotide phosphorylase is an important enzyme in the growth phase adaptation to stationary phase
-
-
?
additional information
?
-
-
examination of phosphorolytic activity. Enzyme is able to digest a substrate with a 3' single-stranded tail as well as a substrate possessing a 3' stem-loop structure. Presence of nucleoside diphosphates has no effect on the phosphorolytic activity
-
-
?
additional information
?
-
under conditions of excess nucleoside diphosphate and low concentrations of phosphate, PNPase catalyses the reverse reaction to add 3' extensions to transcripts
-
-
?
additional information
?
-
-
under conditions of excess nucleoside diphosphate and low concentrations of phosphate, PNPase catalyses the reverse reaction to add 3' extensions to transcripts
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
either in the form of a homotrimeric enzyme or associated in a multiprotein complex, the degradosome, PNPase is involved in RNA processing
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase and RNAse II play an essential role in degrading fragments of mRNA generated by prior cleavage by endonucleases
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase accounts for 10% of total mRNA decay, PNPase can bind double stranded DNA, however the affinity is lower than that obtained for both RNA and single stranded DNA binding
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase synthesizes long, highly heteropolymeric poly(A) tails in vivo and accounts for all of the residual polyadenylylation in poly(A) polymerase deficient strains, in addition PNPase is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing wild-type cultures
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase exonuclease activity plays an essential role in tRNA, mRNA and ribosome metabolism
-
-
?
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase is involved in RNA degradation
-
r
RNAn + a nucleoside diphosphate
RNAn+1 + phosphate
-
PNPase specifically binds to 8-oxoguanine-containing RNA, it is suggested that PNPase discriminate between oxidized and normal RNA which my contribute to a high fidelity of translation
-
-
?
RNAn+1 + phosphate
RNAn + a nucleoside diphosphate
-
in addition to its degradative role, PNPase can also function as a polymerase, adding 3' tails to transcripts. The reverse of degradation is favored when nucleoside diphosphate rather than inorganic phosphate is present in excess
-
-
r
additional information
?
-
regulates its own expression at the level of mRNA stability and translation
-
-
?
additional information
?
-
-
regulates its own expression at the level of mRNA stability and translation
-
-
?
additional information
?
-
-
suppression of Rho-dependent transcription termination within the enzyme gene and its restoration by enzyme protein is an autogenous regulation circuit that modulates enzyme gene expression during cold acclimation
-
-
?
additional information
?
-
-
polynucleotide phosphorylase is essential for growth at low temperatures, while polymerization activity is not essential. RNase PH domains 1 and 2 of polynucleotide phosphorylase are important for its cold shock function, suggesting that the RNase activity of the enzyme is critical for its essential function at low temperature. Its polymerization activity is dispensable in its cold shock function. The RNase R , which is cold inducible, cannot complement the cold shock function of PNPase
-
-
?
additional information
?
-
-
polyribonucleotide phosphorylase-mediated degradation is a major regulatory event controlling the levels of sRNAs, namely stationary phase regulators MicA and RybB, that are required for the accurate expression of outer membrane proteins. Degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. Polyribonucleotide phosphorylase is an important enzyme in the growth phase adaptation to stationary phase
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
-
no effect in E. coli enzyme, 0.005 mM, 3fold activation of Bacillus stearothermophilus enzyme
Mg2+
Mn2+
Mn2+
-
200000 Da form requires Mn2+ for NDP polymerization, polymerization of GDP proceedes efficiently in presence of Mn2+ at 60°C, polymerization with a mutant enzyme from E. coli Q13 requires Mn2+ rather than Mg2+
Mn2+
Mn2+ can substitute for Mg2+ as an essential co-factor for PNPase catalysis
Mn2+
-
manganese can substitute for magnesium as the catalytic metal in PNPase, and RNA degradation
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
-
5 mM, 28°C, presence of MgCl2, 30% residual activity. Allosteric inhibition of both polymerization and phosphorolytic activities, mixed-type inhibition toward phosphate
citrate
-
a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. PNPase-dependent cells show reduced growth in the presence of increased citrate concentration. In vitro, citrate directly binds and modulates PNPase activity, and the enzyme is inhibited by binding of metal-chelated citrate, predominantly complexed as magnesium-citrate, in the active site at physiological concentrations. In the contrary, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
citrate
-
a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. PNPase-dependent cells show reduced growth in the presence of increased citrate concentration. In vitro, citrate directly binds and modulates PNPase activity, and the enzyme is inhibited by binding of metal-chelated citrate, predominantly complexed as magnesium-citrate, in the active site at physiological concentrations. In the contrary, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity, this vestigial site as an allosteric binding pocket that recognizes metal-free citrate
diphosphate
-
activation of PNPase RNA synthesis activity at very low concentrations of phosphate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000166
globin mRNA
-
pH 8.0, 37°C, phosphorolysis of poly(A)tail from rabbit globin mRNA
-
additional information
additional information
-
values for Km with polynucleotides longer than 20 nucleotides are much smaller than the Km for oligonucleotides which lies at approx. 0.05 mM
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
recombinant enzyme activity is measured as repression of beta-galactosidase activity in the reporter expression sytem, RNA binding affinity and in vitro RNA-binding activities of wild-type and mutant enzymes, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
intracellular levels of PNPase are regulated by polyadenylation levels of transcripts
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
evolution
-
polynucleotide phosphorylase is a conserved, widely distributed phosphorolytic 3'-5' exoribonuclease
malfunction
metabolism
physiological function
additional information
physiological function
polynucleotide phosphorylase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. They facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. Polynucleotide phosphorylase may contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responds differently to the absence or presence of PNPase and RNase PH. Polynucleotide phosphorylase and RNase PH stabilize only Hfq-bound sRNAs
physiological function
polynucleotide phosphorylase contributes to the degradation of specific short mRNA fragments, the majority of which bind RNA chaperone Hfq and are derived from targets of sRNAs. The mRNA-derived fragments accumulate in the absence of polynucleotide phosphorylase or its exoribonuclease activity and interact with polynucleotide phosphorylase. Mutations in chaperone Hfq or in the seed pairing region of some sRNAs eliminate the requirement of polynucleotide phosphorylase for their stability
malfunction
-
deletion of the pnp gene, encoding polynucleotide phosphorylase, results in increased biofilm formation in Escherichia coli
malfunction
-
loss-of-function mutations in pnp result in a decreased stability of several sRNAs including RyhB, SgrS, and CyaR and also decrease both the negative and positive regulation by sRNAs. The defect in stability of CyaR and in negative and positive regulation are suppressed by deletion mutations in RNase E. Lack of sRNA-mediated regulation in the absence of an active form of PNPase is due to the rapid turnover of sRNA resulting from an increase in RNase E activity and/or an increase in access of other ribonucleases to sRNAs. The defect in sRNA regulation caused by the pnp mutations is independent of Hfq. While Hfq does not appear to be limiting, it seems possible that lack of PNPase leads to inactivation of Hfq
malfunction
-
spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair system, are sharply reduced in a polynucleotide phosphorylase-deficient Escherichia coli strain
metabolism
-
PNPase, together with the endonuclease RNase E, the DEAD-box RNA helicase RhlB, and enolase, constitutes the RNA degradosome, a multiprotein machine devoted to RNA degradation
metabolism
-
the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity, a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome and on global gene expression, detailed overview
physiological function
PNPase is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors
physiological function
-
metabolite-bound PNPase structure and evidence for an allosteric pocket, overview
physiological function
-
polynucleotide phosphorylase is an RNA processing enzyme and a component of the RNA degradosome. It plays an important role in RNA processing and turnover, being implicated in RNA degradation and in polymerization of heteropolymeric tails at the 3'-end of mRNA. PNPase is necessary to maintain bacterial cells in the planktonic mode through downregulation of pgaABCD expression and poly-N-acetylglucosamine production. But the pnp gene is not essential. Negative regulation of the poly-N-acetylglucosamine biosynthetic operon pgaABCD by PNPase
physiological function
-
polynucleotide phosphorylase plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates that can be converted to deoxyribonucleoside diphosphates by ribonucleotide reductase
physiological function
-
in vitro, enzyme forms a ternary complex comprised of PNPase, chaperine Hfq, and sRNA and PNPase and Hfq may also form a ribonucleoprotein complex in the cell. In in vitro studies, PNPase readily degrades sRNAs in the absence of Hfq, but binds and is unable to degrade sRNAs in its presence
physiological function
-
polynucleotide phosphorylase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by radiomimetic zeocin. Homologous recombination does not require polynucleotide phosphorylasephosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on polynucleotide phosphorylase catalytic activity and cannot be suppressed by overexpression of RNase II. Polynucleotide phosphorylase mutants are more sensitive than the wild-type to zeocin. This phenotype depends on polynucleotide phosphorylasephosphorolytic activity and is suppressed by RNase II
additional information
-
the increase in the rNDP pools generated by polynucleotide phosphorylase degradation of RNA is responsible for the spontaneous mutations observed in an mismatch repair-deficient background, and is also responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine
additional information
-
the S1 and KH domains of polynucleotide phosphorylase determine the efficiency of RNA binding and enzyme autoregulation, modeling of the roles of the KH and S1 domains in PNPase-RNA interactions and in substrate binding, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100000
-
low molecular weight form catalyzing phosphorolysis but unable to catalyze the polymerization of NDP's, can only phosphorolyze short-chain polymers and requires higher Mg2+ ion concentration
200000
-
this form requires Mn2+ for NDP polymerization and has a higher Km for poly(A) phosphorolysis
48000
-
alpha3,beta2 or alpha3,betan, x * 86000 + x * 48000, enzyme form B is obtained by keeping the ionic strength at 200 mM during purification on Sephadex G-200, at lower salt concentrations the beta subunit tends to dissociate and the enzyme reverts to the A form
86000
additional information
-
PNPase, the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome
86000
-
alpha3,beta2 or alpha3,betan, x * 86000 + x * 48000, enzyme form B is obtained by keeping the ionic strength at 200 mM during purification on Sephadex G-200, at lower salt concentrations the beta subunit tends to dissociate and the enzyme reverts to the A form
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
alpha3,beta2 or alpha3,betan, x * 86000 + x * 48000, enzyme form B is obtained by keeping the ionic strength at 200 mM during purification on Sephadex G-200, at lower salt concentrations the beta subunit tends to dissociate and the enzyme reverts to the A form
monomer
-
domain organization of the enzyme monomer, homology modeling with KH domain and S1 domain, overview
trimer
additional information
trimer
-
alpha3, 3 * 84000-95000, enzyme form A, ultrastructural observations
additional information
enzyme domains within residues 158-262 and 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration
additional information
-
enzyme domains within residues 158-262 and 473-577 contain interaction sites for RNase E within a betabetaalphabetabetaalpha domain configuration
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
three-dimensional modeling of enzyme based on Streptomyces antibioticus protein structure. The binding domain for RNase E is located on the monomer surface, facing outward from the trimeric tertiary structure
wild-type and C-terminal KH/S1 domain truncated mutant at resolutions of 2.6 and 2.8 A, respectively. The six PH domains assemble into a ring-like structure containing a central channel
PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA, hanging droplet vapor diffusion method, crystals for the PNPase core/RNase E micro-domain crystals are grown using 0.2 M ammonium nitrate, and 20% (w/v) PEG 3350, crystals for the PNPase core/RNase E microdomain-RNA complex are produced using 0.2 M diammonium hydrogen citrate, and 17% PEG 3350. The optimal reservoir buffer for the PNPase core/RNase E micro-domain-RNA-tungstate crystals is composed of 0.2 M di-ammonium hydrogen citrate, 17% PEG 3350, about pH 4.5, 50 mM disodium tungstate. Crystals for the PNPase core/RNase E micro-domain-Mn2+ co-crystals are prepared using 2.5 M NaCl, 9% (w/v) PEG 6000, 20 mM sodium citrate, and 20 mM manganese acetate tetrahydrate
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A552T
complementation of growth defect at 15°C of host strain. Modest effect of mutation on phosphorolytic activity and protein abundance
E371K
complementation of growth defect at 15°C of host strain. Modest effect of mutation on phosphorolytic activity and protein abundance
E81D
complementation of growth defect at 15°C of host strain. Increase in PNPase abundance without significantly impairing phosphorolytic activity
E81K
complementation of growth defect at 15°C of host strain. Increase in PNPase abundance without significantly impairing phosphorolytic activity
P98L
complementation of growth defect at 15°C of host strain, forms of smaller colonies than host strain. Severe reduction of enzyme activity and increased PNPase expression levels
R97C
complementation of growth defect at 15°C of host strain. Severe reduction of enzyme activity and increased PNPase expression levels
V521I
complementation of growth defect at 15°C of host strain. Modest effect of mutation on phosphorolytic activity and protein abundance
V639D
complementation of growth defect at 15°C of host strain, migrates slower than wild-type on SDS-PAGE, forms of smaller colonies than host strain. Increase in PNPase abundance without significantly impairing phosphorolytic activity
A552T
ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.7, as compared with 1.0 in wild-type
E371K
ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.6, as compared with 1.0 in wild-type
E81D
impaired growth at 15°C, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 1.6, as compared with 1.0 in wild-type
E81K
impaired growth at 15°C, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.4, as compared with 1.0 in wild-type
F635A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme
F635A/F638A/H650A
-
site-directed mutagenesis, the mutant enzyme shows highly reduced activity and an increased RNA binding constant compared to the wild-type enzyme
F635R/F638R/H650R
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
F638A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
G570C
-
site-directed mutagenesis, the mutant enzyme shows highly reduced activity and an increased RNA binding constant compared to the wild-type enzyme
G570C/V679A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme
H650A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme
I555T
-
site-directed mutagenesis, the mutant enzyme shows slightly reduced activity compared to the wild-type enzyme
I576A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
I576A/F638A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme
I576T
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
I576T/F638A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
I576T/T585A
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type enzyme
K571L
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
K571Q
-
site-directed mutagenesis, the mutant enzyme shows reduced activity and an increased RNA binding constant compared to the wild-type enzyme
P98L
impaired growth at 15°C, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.03, as compared with 1.0 in wild-type
R83A
the mutation has little apparent effect on activity but causes the full-length PNPase to stall on RNA oligomers shorter than eight nucleotides
R97C
ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.1, as compared with 1.0 in wild-type
V304A/V305D
ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.02, as compared with 1.0 in wild-type
V521I
ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.5, as compared with 1.0 in wild-type
V639D
impaired growth at 15°C, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.6, as compared with 1.0 in wild-type
additional information
additional information
construction of hybrid proteins by replacing the S1 RNA binding domain of RNase II for the S1 from enzyme. PNPase S1 domain can partially restore the RNA-binding ability and exonucleolytic activity of Rnase II and is able to induce the trimerization of the Rnase II-PNPase hybrid protein
additional information
-
construction of hybrid proteins by replacing the S1 RNA binding domain of RNase II for the S1 from enzyme. PNPase S1 domain can partially restore the RNA-binding ability and exonucleolytic activity of Rnase II and is able to induce the trimerization of the Rnase II-PNPase hybrid protein
additional information
C-terminal KH/S1 domain truncated mutant, crystallization data. Mutant binds and cleaves RNA less efficiently with an 8fold reduced binding affinity and forms a less stable trimer. Mutation of Arg-residues in the central channel neck region produces defective enzymes that either bind and cleave RNA less efficiently or generate longer cleaved oligonucleotide products
additional information
-
C-terminal KH/S1 domain truncated mutant, crystallization data. Mutant binds and cleaves RNA less efficiently with an 8fold reduced binding affinity and forms a less stable trimer. Mutation of Arg-residues in the central channel neck region produces defective enzymes that either bind and cleave RNA less efficiently or generate longer cleaved oligonucleotide products
additional information
several new pnp alleles constructed. To identify specific cis-acting determinants of PNPase autoregulation and discriminate between the two proposed models, several pnpL DELTApnp-871 mutants and one DELTApnp-1010t DELTApnp-871 chromosomal double mutant are constructed
additional information
-
several new pnp alleles constructed. To identify specific cis-acting determinants of PNPase autoregulation and discriminate between the two proposed models, several pnpL DELTApnp-871 mutants and one DELTApnp-1010t DELTApnp-871 chromosomal double mutant are constructed
additional information
study on the effect of specific mutations in the two RNA binding domains KH and S1. Removal of critical motifs that stabilize the hydrophobic core of each domain, as well as a complete deletion of both severely impaireds binding to RNA. all mutants are enzymatically active but display significant changes in the kinetic behaviour of both phosphorolysis and polymerization activities. Mutants do not autoregulate efficiently and are unable to complement the growth defect of a chromosomal enzmye deletion at 18°C
additional information
-
study on the effect of specific mutations in the two RNA binding domains KH and S1. Removal of critical motifs that stabilize the hydrophobic core of each domain, as well as a complete deletion of both severely impaireds binding to RNA. all mutants are enzymatically active but display significant changes in the kinetic behaviour of both phosphorolysis and polymerization activities. Mutants do not autoregulate efficiently and are unable to complement the growth defect of a chromosomal enzmye deletion at 18°C
additional information
-
deletion of KHS1 domain, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.6, as compared with 1.0 in wild-type. Deletion mutant lacking amino acids 549-709, no growth at 15°C. Both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold
additional information
deletion of KHS1 domain, ratio of phosphorolytic activity to polynucleotide phosphorylase activity 0.6, as compared with 1.0 in wild-type. Deletion mutant lacking amino acids 549-709, no growth at 15°C. Both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold
additional information
-
in a mutant lacking polyribonucleotide phosphorylase activity, the pattern of outer membrane proteins is changed. In stationary phase, stationary phase regulator MicA RNA levels are increased in the mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein
additional information
-
construction of a strain in which PNPase activity is uncoupled from the degradosome through the deletion of the C-terminal degradosome-scaffold-ing domain of RNase E. Compared with the parental strain, significant differences are distributed across many metabolic pathways, including the Krebs cycle, amino acid synthesis, and glycolysis in the mutant strain. Salient differences are seen for amino acids and increases in the concentrations of succinate, fumarate, and malate, suggesting uncoupling of the two halves of the Krebs cycle
additional information
-
construction of domain deletion mutants DELTAKH DELTAS1, DELTAKH, and DELTAS1
additional information
-
deletion of gene pnp in Eschericchia coli strain C-1a leading to strong cell aggregation in liquid medium dependent on the extracellular polysaccharide poly-N-acetylglucosamine. Operon pgaABCD transcript levels are increased in the pnp mutant compared to the wild-type enzyme. Inactivation of the pnp gene induces poly-N-acetylglucosamine production. The aggregative phenotype of the C-5691 (DELTApnp) strain is complemented by basal expression from a multicopy plasmid of the pnp gene under araBp promoter, overview
additional information
-
genetic selection and screen for mutants defective in the post-transcriptional regulation of gene expression by sRNAs, i.e. CyaR, SgrS, and RyhB. Each of the pnp mutations isolated, as well as a pnp deletion, are transduced into strain DJ624, overview. The defect in sRNA regulation caused by the pnp mutations is independent of Hfq. While Hfq does not appear to be limiting, it seems possible that lack of PNPase leads to inactivation of Hfq
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
stabilized against heat inactivation by the presence of NDP's but not by NMP's, NTP's, DNA or substrate oligonucleotides with free 3'-OH termini
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, Q-Sepharose column chromatography, and Mono-Q column chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain ENS134-3
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of enzyme mutant R153A/R372A/R405A/R409A in Escherichia coli strain BL21(DE3)
-
expression of wild-type and mutant enzymes in Escherichia coli strain ENS134-3 from modofied plasmid pAW101 and in beta-galactosidase reporter strain IBPC7322(lambdaGF2), overview
-
overexpression in Escherichia coli in the presence or absence of increased levels of polyadenylated transcripts
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
PNPase regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
after heating at 100°C for 1 min 25-30% of the original activity can be recovered by dissolving the precipitate in 6 M guanidine-HCl followed by dialysis
-
refolding of SDS-PAGE purified PNPase diluted 50fold into enzyme buffer containing 0.5% Triton X-100 and 0.5 mg/ml bovine serum albumin
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Zhou, Z.; Deutscher, M.P.
An essential function for the phosphate-dependent exoribonucleases RNase PH and polynucleotide phosphorylase
J. Bacteriol.
179
4391-4395
1997
Escherichia coli
Littauer, U.Z.; Soreq, H.
Polynucleotide phosphorylase
The Enzymes, 3rd. Ed. (Boyer, P. D. , ed. )
15B
517-553
1982
Azotobacter vinelandii, Achromobacter sp., Synechococcus elongatus PCC 7942 = FACHB-805, Geobacillus stearothermophilus, Bacillus amyloliquefaciens, Brevibacterium sp., Cavia porcellus, Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Halobacterium salinarum, Micrococcus luteus, Nicotiana tabacum, Rattus norvegicus, Sinorhizobium meliloti, Rhodospirillum rubrum, Streptococcus pyogenes, Thermus aquaticus, Bacillus amyloliquefaciens BaM-2, Achromobacter sp. KR. 170-4
-
Godefroy-Colburn, T.; Grunenberg-Manago, M.
Polynucleotide phosphorylase
The Enzymes, 3rd. Ed. (Boyer, P. D. , ed. )
7
533-574
1972
Ascaris lumbricoides, Auxenochlorella pyrenoidosa, Cavia porcellus, Escherichia coli, Halobacterium salinarum, Homo sapiens, Lactiplantibacillus plantarum, Micrococcus luteus, Neisseria meningitidis, Pseudomonas aeruginosa, Rattus norvegicus, Salmonella enterica subsp. enterica serovar Typhimurium, Spinacia oleracea, Synechococcus elongatus PCC 7942 = FACHB-805, Triticum aestivum
-
Soreq, H.; Littauer, U.Z.
Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3 sequences of RNA
J. Biol. Chem.
252
6885-6888
1977
Escherichia coli
Smith, J.C.; Eaton, M.A.W.
Purification of polynucleotide phosphorylase by affinity chromatography and some properties of the purified enzymes
Nucleic Acids Res.
1
1763-1773
1974
Geobacillus stearothermophilus, Escherichia coli
Carpousis, A.J.; Van Houwe, G.; Ehretsmann, C.; Krisch, H.M.
Copurification of E. coli RNase E and PNPase: evidence for a specific association between two enzymes important in RNA processing and degradation
Cell
76
889-900
1994
Escherichia coli
Lisitsky, I.; Schuster, G.
Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase
Eur. J. Biochem.
261
468-474
1999
Escherichia coli
Mohanty, B.K.; Kushner, S.R.
Polynucleotide phosphorylase functions both as a 3' -> 5' exonuclease and a poly(A) polymerase in Escherichia coli
Proc. Natl. Acad. Sci. USA
97
11966-11971
2000
Escherichia coli
Spickler, C.; Mackie, G.A.
Action of RNase II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure
J. Bacteriol.
182
2422-2427
2000
Escherichia coli
Zangrossi, S.; Briani, F.; Ghisotti, D.; Regonesi, M.E.; Tortora, P.; Deho, G.
Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli
Mol. Microbiol.
36
1470-1480
2000
Escherichia coli
Baginsky, S.; Shteiman-Kotler, A.; Liveanu, V.; Yehudai-Resheff, S.; Bellaoui, M.; Settlage, R.E.; Shabanowitz, J.; Hunt, D.F.; Schuster, G.; Gruissem, W.
Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome
RNA
7
1464-1475
2001
Escherichia coli, Spinacia oleracea
Hayakawa, H.; Kuwano, M.; Sekiguchi, M.
Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein
Biochemistry
40
9977-9982
2001
Escherichia coli
Yehudai-Resheff, S.; Hirsh, M.; Schuster, G.
Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts
Mol. Cell. Biol.
21
5408-5416
2001
Escherichia coli, Spinacia oleracea
Bermudez-Cruz, R.M.; Garcia-Mena, J.; Montanez, C.
Polynucleotide phosphorylase binds to ssRNA with same affinity as to ssDNA
Biochimie
84
321-328
2002
Escherichia coli
Mohanty, B.K.; Kushner, S.R.
Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E
Mol. Microbiol.
45
1315-1324
2002
Escherichia coli
Duran-Figueroa, N.V.; Pina-Escobedo, A.; Schroeder, I.; Simons, R.W.; Garcia-Mena, J.
Polynucleotide phosphorylase interacts with ribonuclease E through a betabetaalphabetabetaalpha domain
Biochimie
88
725-735
2006
Escherichia coli (P05055), Escherichia coli
Briani, F.; Del Favero, M.; Capizzuto, R.; Consonni, C.; Zangrossi, S.; Greco, C.; De Gioia, L.; Tortora, P.; Deho, G.
Genetic analysis of polynucleotide phosphorylase structure and functions
Biochimie
89
145-157
2007
Escherichia coli, Escherichia coli (P05055)
Marchi, P.; Longhi, V.; Zangrossi, S.; Gaetani, E.; Briani, F.; Deho, G.
Autogenous regulation of Escherichia coli polynucleotide phosphorylase during cold acclimation by transcription termination and antitermination
Mol. Genet. Genomics
278
75-84
2007
Escherichia coli
Amblar, M.; Barbas, A.; Gomez-Puertas, P.; Arraiano, C.M.
The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization
RNA
13
317-327
2007
Escherichia coli (P05055), Escherichia coli
Shi, Z.; Yang, W.Z.; Lin-Chao, S.; Chak, K.F.; Yuan, H.S.
Crystal structure of Escherichia coli PNPase: Central channel residues are involved in processive RNA degradation
RNA
14
2361-2371
2008
Escherichia coli (P05055), Escherichia coli
Matus-Ortega, M.E.; Regonesi, M.E.; Pina-Escobedo, A.; Tortora, P.; Deho, G.; Garcia-Mena, J.
The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature
Biochim. Biophys. Acta
1769
194-203
2007
Escherichia coli (P05055), Escherichia coli
Awano, N.; Inouye, M.; Phadtare, S.
RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II
J. Bacteriol.
190
5924-5933
2008
Escherichia coli, Escherichia coli JM83
Chang, S.A.; Cozad, M.; Mackie, G.A.; Jones, G.H.
Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates
J. Bacteriol.
190
98-106
2008
Escherichia coli, Streptomyces coelicolor
Carzaniga, T.; Briani, F.; Zangrossi, S.; Merlino, G.; Marchi, P.; Deho, G.
Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited
J. Bacteriol.
191
1738-1748
2009
Escherichia coli (P05055), Escherichia coli
Del Favero, M.; Mazzantini, E.; Briani, F.; Zangrossi, S.; Tortora, P.; Deho, G.
Regulation of Escherichia coli polynucleotide phosphorylase by ATP
J. Biol. Chem.
283
27355-27359
2008
Escherichia coli
Nurmohamed, S.; Vaidialingam, B.; Callaghan, A.J.; Luisi, B.F.
Crystal structure of Escherichia coli polynucleotide phosphorylase core bound to RNase E, RNA and manganese: Implications for catalytic mechanism and RNA degradosome assembly
J. Mol. Biol.
389
17-33
2009
Escherichia coli (A7ZS61), Escherichia coli
Andrade, J.M.; Arraiano, C.M.
PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins
RNA
14
543-551
2008
Escherichia coli, Escherichia coli MG1693
Carzaniga, T.; Antoniani, D.; Deho, G.; Briani, F.; Landini, P.
The RNA processing enzyme polynucleotide phosphorylase negatively controls biofilm formation by repressing poly-N-acetylglucosamine (PNAG) production in Escherichia coli C
BMC Microbiol.
12
270
2012
Escherichia coli, Escherichia coli MG1655
Becket, E.; Tse, L.; Yung, M.; Cosico, A.; Miller, J.H.
Polynucleotide phosphorylase plays an important role in the generation of spontaneous mutations in Escherichia coli
J. Bacteriol.
194
5613-5620
2012
Escherichia coli
Wong, A.G.; McBurney, K.L.; Thompson, K.J.; Stickney, L.M.; Mackie, G.A.
The S1 and KH domains of polynucleotide phosphorylase determine the efficiency of RNA binding and autoregulation
J. Bacteriol.
195
2021-2031
2013
Escherichia coli
Nurmohamed, S.; Vincent, H.A.; Titman, C.M.; Chandran, V.; Pears, M.R.; Du, D.; Griffin, J.L.; Callaghan, A.J.; Luisi, B.F.
Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli
J. Biol. Chem.
286
14315-14323
2011
Escherichia coli, Escherichia coli MG1655
De Lay, N.; Gottesman, S.
Role of polynucleotide phosphorylase in sRNA function in Escherichia coli
RNA
17
1172-1189
2011
Escherichia coli, Escherichia coli MG1193
Carzaniga, T.; Deho, G.; Briani, F.
RNase III-independent autogenous regulation of Escherichia coli polynucleotide phosphorylase via translational repression
J. Bacteriol.
197
1931-1938
2015
Escherichia coli
Bandyra, K.J.; Sinha, D.; Syrjanen, J.; Luisi, B.F.; De Lay, N.R.
The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes
RNA
22
360-372
2016
Escherichia coli
Carzaniga, T.; Sbarufatti, G.; Briani, F.; Deho, G.
Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli
BMC Microbiol.
17
81
2017
Escherichia coli
Cameron, T.A.; De Lay, N.R.
The phosphorolytic exoribonucleases polynucleotide phosphorylase and RNase PH stabilize sRNAs and facilitate regulation of their mRNA targets
J. Bacteriol.
198
3309-3317
2016
Escherichia coli (P05055), Escherichia coli
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