Information on EC 3.1.13.4 - poly(A)-specific ribonuclease

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.1.13.4
-
RECOMMENDED NAME
GeneOntology No.
poly(A)-specific ribonuclease
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
exonucleolytic cleavage of poly(A) to 5'-AMP
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
deadenylation
-
-
hydrolysis of phosphoric ester
CAS REGISTRY NUMBER
COMMENTARY hide
110541-21-4
-
215797-47-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain BL21 (DE3)
-
-
Manually annotated by BRENDA team
genes PARN-1, PARN-2, and PARN-3
-
-
Manually annotated by BRENDA team
genes PARN-1, PARN-2, and PARN-3
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(A)12 + H2O
AMP
show the reaction diagram
-
full degradation of substrate
-
?
(A)6 + H2O
5'-AMP + ?
show the reaction diagram
-
-
-
-
?
2',5'-linked oligoadenylates + H2O
5'-AMP
show the reaction diagram
-
in interferon treated cells
-
-
?
3',5-linked oligoadenylates + H2O
5'-AMP
show the reaction diagram
AAAACAAAAA + H2O
AMP + ?
show the reaction diagram
-
plus a further degradation product containing four A nucleotides and one C nucleotide
-
?
MFA2pG mRNA + H2O
?
show the reaction diagram
-
-
-
-
?
poly(A) + H2O
5'-AMP + ?
show the reaction diagram
-
average substrate length about 200 A
-
-
?
poly(A) + H2O
AMP
show the reaction diagram
poly(A) + H2O
AMP + ?
show the reaction diagram
-
no accumulation of polynucleotides
-
?
poly(A) RNA + H2O
5'-AMP + ?
show the reaction diagram
poly(A)-mRNA + H2O
5'-AMP
show the reaction diagram
poly(A)-poly(U) + H2O
5'-AMP + ?
show the reaction diagram
poly(a)-ssDNA + H2O
5'-dAMP
show the reaction diagram
-
-
-
?
polyadenylated RNA containing AU-rich elements + H2O
RNA containing AU-rich elements + AMP
show the reaction diagram
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
poly(A) RNA + H2O
5'-AMP + ?
show the reaction diagram
poly(A)-mRNA + H2O
5'-AMP
show the reaction diagram
Q8VDG3
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
-
activates; less effective than Mg2+ or Mn2+
Ca2+
-
activates; less effective than Mg2+ or Mn2+
Cd2+
-
2 mM, activates reaction, can modulated the substrate length requirement
Cu2+
-
activates; less effective than Mg2+ or Mn2+
Fe2+
-
can substitute for Mg2+, Mn2+, optimal at 0.01 mM, two binding sites
Sn2+
-
activates; less effective than Mg2+ or Mn2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl) cytosine
-
-
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N4-benzoyl cytosine
-
-
3-aminotriazole
-
competitive inhibitor
9-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N6-benzoyl adenine
-
-
ADP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
ATP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
Ca2+
-
3 mM gradually decreases activity about 15fold
cap analogue 7-methylguanosine(5')ppp(5')G
-
allosteric inhibitor of PARN in the presence of a physiological K+ concentration
CH3COOK
Co2+
-
3 mM gradually decreases activity about 78fold
deoxyATP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
deoxyGTP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
EDTA
activity is completely lost when the ions are removed from the wild-type enzyme by incubation with 10mM
eIF4E
-
competition between PARN and eIF4E for the 5'-cap
-
GDP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
GMP
-
purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
GTP
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors, furthermore PARN does not discriminate whether there is ribose or deoxyribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
guanidine hydrochloride
-
full-length enzyme retains most of its activity at guanidine hydrochloride concentrations below 0.5 M, whereas an abrupt decrease of the activity of p54 and p46 is found when guanidine hydrochloride concentration is increased
K3PO4
kanamycin A
-
50% inhibition by high nanomolar concentrations, competition with divalent metal ions
kanamycin B
-
50% inhibition by high nanomolar concentrations, competition with divalent metal ions
lividomycin
-
50% inhibition by high nanomolar concentrations, competition with divalent metal ions
m7G(5')ppp(5')G cap analogue
-
-
Mg2+
-
behaves as a destabilizer of the overall structural stability of PARN, promotes thermal unfolding and aggregation at high temperatures
Mn2+
-
3 mM gradually decreases activity about 5fold
N-ethylmaleimide
-
-
NaCl
-
inhibition at 0.2 M ionic strength; inhibits at 150-200 mM
neomycin
-
antibiotic neomycin B inhibits approximately 80% of PARN activity at a concentration of 10 microg/ml
neomycin B
-
50% inhibition by high nanomolar concentrations, inhibition is pH-dependent, competition with divalent metal ions
o-Iodobenzoate
-
-
paromomycin
-
50% inhibition by high nanomolar concentrations, competition with divalent metal ions
phosphate
-
inhibits at 10 mM
Poly(A)
; strong inhibition of both spermidine- and polyadenylate-binding protein-activated hPAN complex
poly(A)-binding protein PAB 1
-
inhibits; phased poly(A) shortening
-
poly(C)
; strong inhibition of spermidine-, slight inhibition of polyadenylate-binding protein-activated hPAN complex
poly[2'-O-(2,4-dinitrophenyl)]poly-(A)
-
competitive
-
purine nucleotides
-
purine triphosphate nucleotides (RTP) behave as non-competitive inhibitors while purine monophosphate (RMP) and diphosphate nucleotides (RDP) exhibit competitive inhibition, furthermore PARN does not discriminate whether there is ribose or deoxy-ribose in the nucleotides, Mg2+ releases the inhibition by RDPs and RTPs, but not by RMPs
-
synthetic fluoro-pyranosyl nucleosides
-
synthetic nucleoside analogues bearing a fluoroglucopyranosyl sugar moiety and benzoyl-modified cytosine or adenine as a base can effectively inhibit human PARN
-
tobramycin
-
50% inhibition by high nanomolar concentrations, competition with divalent metal ions
Zn2+
enzyme is inhibited by high zinc concentration with only two ions and 1mM Zn2+
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12-tetradecanoyl phorbol 13-acetate
induces Noc transcript levels in quiescent NIH3T3 cultures
3'untranslated regions of RNA
-
regulates RNase acitvity
-
CH3COOK
K+
-
allosteric activator of PARN and its truncated forms, optimal concentration is around 100 mM, with the increase of the K+ concentration, the enzyme reaches its Vmax at a much lower substrate concentration
Pan2 protein
-
required for poly(A)-binding in poly(A) cleavage
-
poly(A)-binding protein PAB 1
-
PAB 1 required
-
polyadenylate-binding protein
or spermidine, required for activity of hPAN complex, cannot stimulate hPAN2 alone
-
spermidine
tristetraprolin
-
required for degradation of polyadenylated RNA containing AU-rich elements
-
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0051
Poly(A)
-
-
0.0000236 - 0.00793
poly(A) RNA
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 7.6
Poly(A)
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.645
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl) cytosine
-
at pH 7.0, 30°C
0.21
1-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N4-benzoyl cytosine
-
at pH 7.0, 30°C
0.51
9-(3',4'-dideoxy-3'-fluoro-beta-D-glucopyranosyl)-N6-benzoyl adenine
-
at pH 7.0, 30°C
0.5
ADP
-
at pH 7.0, 30°C
1.6
AMP
-
at pH 7.0, 30°C
3.2
ATP
-
at pH 7.0, 30°C
0.7
CMP
-
at pH 7.0, 30°C
2.8
deoxyATP
-
at pH 7.0, 30°C
2
deoxyGTP
-
at pH 7.0, 30°C
2.8
GDP
-
at pH 7.0, 30°C
4.5
GMP
-
at pH 7.0, 30°C
3.3
GTP
-
at pH 7.0, 30°C
0.0004
neomycin B
-
pH 7.0
0.00007
Poly(A)
37°C, pH 7.5, spermidine-activated hPAN
0.002
poly(C)
37°C, pH 7.5, spermidine-activated hPAN
0.098
poly[2'-O-(2,4-dinitrophenyl)]poly-(A)
-
pH not specified in the publication, temperature not specified in the publication
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0625
-
-
211000
-
pH 7.0, 30°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
-
assay at
7.6
-
assay at
8.7 - 9.4
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.4 - 10.5
-
pH 8.4: about 25% of maximum activity, pH 10.5: about 15% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
Hep G2 cells
Manually annotated by BRENDA team
-
mouse macrophage cell line
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
Pab1p contains a non-essential leucine-rich nuclear export signal and shuttles in an Xpo1/Gsp-1-dependent manner between the nucleus and the cytoplasm
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719)
Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719)
Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)
Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)
Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)
Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)
Rhodobacter sphaeroides (strain ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158)
Rhodobacter sphaeroides (strain ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
Schizosaccharomyces pombe (strain 972 / ATCC 24843)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17000
2 * 17000, SDS-PAGE, dimerization of enzyme fragment PARN(443-560)
18500
2 * 18500, calculated
31000
-
1 * 58000, 1 * 31000, SDS-PAGE
37000
dimerization of enzyme fragment PARN(443-560), gel filtration
38460
gel filtration coupled with multiangle light scattering
42000
immunoblotting
45000
-
x * 45000, recombinant CNOT6L nuclease domain, SDS-PAGE
46000
-
truncated form completely missing the RRM
58000
-
1 * 58000, 1 * 31000, SDS-PAGE
62000 - 74000
-
-
62000
-
PARN (p62), residues 1-540
73500
-
amino acid analysis
75886
x * 135007, catalytic subunit hPAN2, + x * 75886, regulatory subunit hPAN3, calculated from amino acid sequence
76200
x * 76200, His-tagged full-length enzyme, SDS-PAGE
80000
-
gel filtration
135007
x * 135007, catalytic subunit hPAN2, + x * 75886, regulatory subunit hPAN3, calculated from amino acid sequence
161000
180000 - 220000
-
gel filtration
290000
-
gel filtration, full length enzyme
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer
monomer
-
gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer
tetramer
-
4 * 74000, calculated. Poly(A)-specific ribonuclease PARN can self-associate into tetramer and high-order oligomers both in vitro and in living cells. PARN oligomerization is triggered by the R3H domain,which leads to the solvent-exposed Trp219 fluorophore to become buried in a solvent-inaccessible microenvironment. The RRM and C-terminal domains are involved in modulating the dissociation rate of the tetrameric PARN. Tetramerization does not affect the catalytic behavior of the full-length PARN and truncated enzymes containing the RRM domain. Tetramerization significantly enhances the catalytic activity and processivity of the truncated form with the removal of the RRM and C-terminal domains
trimer
-
3 * 74000, SDS-PAGE, a 54000 Da polypeptide fragment is responsible for activity
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the PARN-RNA-recognition motif domain (residues 445-540) with a bound 7-methylguanosine triphosphate nucleotide shows a remarkable conformational flexibility of the RNA-recognition motif domain, crystal structure is refined at a resolution of 2.1 A, protein folds into a three-stranded antiparallel beta-sheet that is flanked by one alpha-helix connecting beta-strands beta1 and beta2
-
free and RNA-bound forms, 20°C, hanging-drop method
-
in silico model for catalytic mechanism and development of a 3D pharmacophore model. Residues Arg99 and Gln109 are involved in the regulation of catalysis. The natural preference of the enzyme for poly(A) is based on favorable biophysical electrostatic and hydrophobic interactions
-
purified recombinant CNOT6L nuclease domain in complex with AMP and poly(A) DNA, hanging drop vapour diffusion method, 0.001 ml of protein solution, containing 15 mg/ml protein and 1 mM DTT, is mixed with 0.001 ml of reservoir solution and equilibrated over 300 ml reservoir solution, containing 0.1 M HEPES, pH 7.5, 1.1 M ammonium tartrate, and 0.2 M NDSB-201, at 16°C, X-ray diffraction structure determination and analysis at 1.94-2.44 A resolution
-
to 2.1 A resolution, one dimer per asymmetric unit
Pop2p crystallized by the sitting drop vapour diffusion technique, to 1.4 A resolution by X-ray crystallography, crystals belong to the space group P212121, contains a fully conserved DEDDh active site
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 55
-
thermal stability and aggregation kinetic parameters, the aggregation is dominated by a first-order kinetics, increase in t0 and the decrease in k implies that Mg2+ can inhibit the rate of enzyme aggregation, overview. Cofactor Mg2+ is also important to PARN stability against inactivation induced by heat treatment, but promotes thermal aggregation at high temperatures. Mg2+ significantly decreases the rate but increases the aggregate size of the 54 kDa wild-type enzyme in a concentration-dependent manner. Effect of mutations on the Mg2+-dependent enzyme thermal aggregation, overview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stabilization with bovine serum albumin
-
stabilization with glycerol and D-galactose
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20oC, 20% glycerol, bovine serum albumin, 2 months, 20% loss of activity
-
-70°C, stable for several months
4°C, 50 mM HEPES buffer, pH 8, 0.01 mM MgOAc, 20 mM KOAc, 20% glycerol, 0.1% igepal
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity purified
by metal affinity resin
-
by Ni2+ affinity chromatography and gel filtration, purity above 98%
-
Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM.
-
His-tag affinity chromatography HiTrap Q HP and 7-Me-GTP-Sepharose affinity chromatography
-
Ni2+ matrix
-
of the recombinant protein
on Ni-NTA resin and by gel filtration, purity above 98%
-
overview on conditions
PARNn purified by glutathione-Sepharose 4B, MonoQ and Superdex 200 gel filtration columns. truncated PARN including the putative cap-binding domain purified by TALON affinity resin. full length PARN purified by Ni-NTA column
-
partial
-
Pop2p purified on Ni2+ column and by gel filtration, to homogeneity
purification of full-length PARN and truncated forms p62 and p54
-
purity above 99%
-
recombinant enzymes
-
recombinant GST-tagged C-terminal 133 amino acids of the PARN-1 from Escherichia coli strain BL21
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
-
recombinant His-tagged full-length enzyme and His-tagged enzyme fragment from Escherichia coli strain BL21(DE3)
recombinant wild-type and mutant E240A, D489A, and H529A CNOT6L nuclease domains from Escherichia coli
-
The wild type and mutant forms of the cap-binding domain of PARN are overexpressed in Escherichia coli BL21(DE3) cells, harvested by centrifugation and suspended in 20 mM Tris-HCl buffer containing 1 M NaCl, 30 mM imidazole, 1 mM 1,4-DL-dithiothreitol, 0.1 mg/ml lysozyme, DNaseI and protease inhibitor cocktail, and lyse with a sonicator. Cell debris and inclusion bodies are removed by centrifugation. The supernatant is loaded on a Ni2+-NTA-agarose column and the proteins are eluted with 20 mM Tris-HCl, 1 M NaCl and 200 mM imidazole.; Unlabeled and [15N], [13C]-labeled PARN cap-binding domains used for NMR experiments are synthesized by the cell-free protein expression system. After reaction, proteins are isolated by Ni2+-affinity chromatography before removing of the His6-tag. Subsequent cation-exchange chromatography yields the purified cap-binding domain.
to homogeneity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 genes, expression and sequence analysis, genotyping of plants, expression of His-tagged wild-type and mutant PARN as functional C-terminal maltose-binding protein-fusion protein in Escherichia coli, expression of wild-type GST-tagged enzyme in Spodoptera frugiperda Sf9 cells using the baculovirus transfection system
-
AHG2, mutant with Arabidopsis abscisic acid hypersensitivity, expression induced by treatment with Arabidopsis abscisic acid, high salinity and osmotic stress
C-terminal-truncated PARN, expression in Escherichia coli
-
cloning and expression of full-length PARN and truncated forms p62 and p54
-
DNA fragment corresponding to amino acids 443-560 in the human PARN sequence cloned into pCR 2.1TOPO vector and subsequently subcloned into the pET19b vector, recombinant PARN or PARN mutants expressed in Escherichia coli strain BL21(DE3)
-
expressed as N-terminal His6-tagged polypeptide in Escherichia coli BL21(DE3)
-
expression in Escherichia coli
-
expression in HEK-293T cells
-
expression in yeast; mutagenized plasmids
-
expression of enzyme subunits in the two-hybrid system using strain L40, expression of lexA-tagged wild-type and mutant Pab1p, Pan2p, and Pan3p in Escherichia coli, expression of full-length and N-terminal constructs of factor Pbp1p in Escherichia coli, overview
-
expression of GFP-tagged wild-type and mutant Pab1p in Xpo1-1 cells at 23°C and at 37°C leading to hyperadenylation of SSA4 mRNA, phenotype or recombinant cells, overview
-
expression of the GST-tagged C-terminal 133 amino acids of the PARN-1 open reading, cloned into the pGEX-6P-1 bacterial expression vector, in Escherichia coli strain BL21
-
expression of wild-type and mutants of the truncated catalytic domain of human CNOT6L, residues 158-555, in Escherichia coli
-
gene PARN, expression of His-tagged enzyme in Escherichia coli strain Bl21(DE3)
-
His tagged, expression in Escherichia coli BL21 (DE3)
-
His-tagged full-length enzyme, comprising residues 1-639, and His-tagged enzyme fragment, PARN(443-560), containing the RNA recognition motif with a 50 amino acids long C-terminal tail, comprising residues 443-560, are expressed in Escherichia coli strain BL21(DE3)
his-tagged recombinant wild type and mutants
-
into pDEST15 vector for Escherichia coli BL21 expression with an N-terminal GST-tag
into vector pET28a, expressed in Escherichia coli BL21(DE3)pLys(S) cells
-
plasmid pET33 containing the human parn gene expressed in Escherichia coli BL21(DE3)
-
R3H domain deletion mutants expressed in Escherichia coli
-
The DNA fragment encoding the cap-binding domain of PARN (residues 430-516) is amplified from a cDNA clone by PCR and is subcloned into pCR2.1. The cap-binding domain is expressed with a His6-tail, a protease cleavage site, a (Gly-Gly-Ser)2-Gly sequence at the N-terminus and a Ser-Gly-Pro-Ser-Ser-Gly sequence at the C-terminus. Deletion mutants of PARN that contain the cap-binding domain and its N- and/or C-terminal flanking regions, encoding residues 420-506, 420-516, 420-536, 430-506, 430-516 and 430-536, are amplified by PCR and subcloned into pET15b. Selected point mutations are introduced into the cap-binding domain (residues 430-516) using a site-directed mutagenesis kit. The wild type and mutant forms of the cap-binding domain of PARN are overexpressed in Escherichia coli.
The PARN fragment comprising amino acids 445-540 is amplified from a human cDNA library and cloned into the expression vector, the glutathione S-transferase-PARN-RNA-recognition motif fusion protein is expressed in Escherichia coli BL21(DE3). PARN mutants are generated from the wild-type clone (pGEX-6P-1 PARN445-540) using a Site-Directed Mutagenesis Kit. SeMet-containing PARN-RNA-recognition motif is expressed in Escherichia coli.
-
wild-type and mutant proteins transformed into Escherichia coli Rosetta (DE3) or Rosetta (DE3) pLysS cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D66A/E68A
-
site-directed mutagenesis, enzymatically inactive PARN, the mutant embryos are retarded, and culminate in an arrest at the bent-cotyledon stage, some show hyperadenylation of transcripts
D28C
-
mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
D292C
-
mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
D382C
-
mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
E30C
-
inactive mutant, acitivity cannot be restored
D28C
-
mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
-
D292C
-
mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
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D382C
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mutants inactive in presence of Mg2+, but with restored activity in presence of soft divalent metal ions, amino acid interacts with catalytically essential metal ions
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E30C
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inactive mutant, acitivity cannot be restored
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D478A
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generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.0 microM
D489A
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site-directed mutagenesis, active site mutant, inactive mutant
E240A
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site-directed mutagenesis, active site mutant, inactive mutant
E455/W456/W475A
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severely defective in cap binding, active deadenylase
E455A
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no defect in its cap binding
F484A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
H529A
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site-directed mutagenesis, active site mutant, inactive mutant
K454A
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generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.03 microM
L197A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
L216A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
L291A
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site-directed mutagenesis, activity and kinetics with RNA substrates compared to the wild-type enzyme
L414A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
M425A
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site-directed mutagenesis, activity and kinetics with RNA substrates compared to the wild-type enzyme
N412A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A/F484A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
P365A/N412A/F484A
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site-directed mutagenesis, active site mutant, not expressable in Escherichia coli
T458A
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generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 30.58 microM
W456A
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cap binding slightly affected
W475A
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severely defective in cap binding
D193A
Mg2+-binding mutant, impaired deadenylase activity
D471A
point mutation is introduced into the cap-binding domain usin site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN
K447A
point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN
K450A
point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN
W468L
point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, mutation significantly decreases the interaction between PARN RNA-recognition motif and the cap analog, the aromatic ring of W468 is directly responsible for the cap recognition and is a functionally critical residue for the cap-binding activity of PARN RNA-recognition motif
H140A
complete loss of activity
A519V
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site-directed mutagenesis of Pab1p, the mutant shows no resistance to 3-AT in contrast to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
D713A
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significant reduction of enzymic activity
D780A
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significant reduction of enzymic activity
E556A
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complete loss of activity, E556 is the key Mg2+-binding residue
G444D
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site-directed mutagenesis of Pab1p, the mutant shows no resistance to 3-AT in contrast to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
G528D
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site-directed mutagenesis of Pab1p, the mutant shows no resistance to 3-AT in contrast to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
H818A
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significant reduction of enzymic activity
R506G
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site-directed mutagenesis of Pab1p, the mutant shows no resistance to 3-AT in contrast to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
V451A
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site-directed mutagenesis of Pab1p, the mutant shows no resistance to 3-AT in contrast to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
Y514C
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site-directed mutagenesis of Pab1p, the mutant shows reduced resistance to 3-AT compared to the wild-type protein, identification of interaction residues and domains with Ppb1p, Pan2p and Pan3p
D50A
has no activity in the presence of 5mM Mg2+
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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leukemic cells from patients with acute lymphoblastic leukemia and acute myeloid leukemia display altered expression for CNOT6, CNOT6L, CNOT7 deadenylase and poly(A)-specific ribonuclease with most significant alterations for poly(A)-specific ribonuclease and CNOT7 mRNA levels. In acute lymphoblastic leukemia, a significant amount of poly(A)-specific ribonuclease is phosphorylated
additional information
Show AA Sequence (999 entries)
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