Information on EC 2.7.7.56 - tRNA nucleotidyltransferase

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

EC NUMBER
COMMENTARY hide
2.7.7.56
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RECOMMENDED NAME
GeneOntology No.
tRNA nucleotidyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
tRNAn+1 + phosphate = tRNAn + a nucleoside diphosphate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tRNA processing
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SYSTEMATIC NAME
IUBMB Comments
tRNA:phosphate nucleotidyltransferase
Brings about the final exonucleolytic trimming of the 3'-terminus of tRNA precursors in Escherichia coli by a phosphorolysis, producing a mature 3'-terminus on tRNA and nucleoside diphosphate. Not identical with EC 2.7.7.8 polyribonucleotide nucleotidyltransferase.
CAS REGISTRY NUMBER
COMMENTARY hide
116412-36-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
full-length protein with translational start at ATG1 (Met1) and two splice variants with translational start at ATG2 (Met6) and ATG3 (Met69)
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Manually annotated by BRENDA team
recombinant
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
genes mth682 and mth683
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Manually annotated by BRENDA team
recombinant enzyme
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Manually annotated by BRENDA team
strain M145
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
additional information
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the core of the exosome is a versatile multisubunit RNA processing enzyme found in archaea and eukaryotes, which includes a ring of six RNase PH subunits, all six RNase PH monomers are catalytically active in the homohexameric RNase PH. Modeling of the Mth exosome RNase PH ring, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
poly(A) + phosphate
?
show the reaction diagram
tRNAn+1 + phosphate
tRNA + a nucleoside diphosphate
show the reaction diagram
tRNAn+1 + phosphate
tRNAn + a nucleoside diphosphate
show the reaction diagram
additional information
?
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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
tRNAn+1 + phosphate
tRNA + a nucleoside diphosphate
show the reaction diagram
tRNAn+1 + phosphate
tRNAn + a nucleoside diphosphate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KCl
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enzyme works optimally at 50 mM KCl, inhibition at 200 mM
N-ethylmaleimide
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p-hydroxymercuribenzoate
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phosphate
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inhibition of the reverse raection
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2
phosphate
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with tRNA-C-C-A-C2 or tRNA-C-C-A-C3, pH 8.0, 37C
0.001
tRNA-C-C-A-C2, tRNA-C-C-A-C3
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pH 8.0, 37C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.14
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formation of CDP from tRNA-C-C-A-Cn
1.6
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phosphorolytic cleavage of poly(A)
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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the core of the exosome is a versatile multisubunit RNA processing enzyme found in archaea and eukaryotes, which includes a ring of six RNase PH subunits
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Manually annotated by BRENDA team
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partial localisation of full-length protein and isoform with translational start at ATG2 (Met6), targeting dependent not only on N-terminal 68 amino acids
Manually annotated by BRENDA team
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partial localisation of full-length protein and isoform with translational start at ATG2 (Met6), targeting dependent not only on N-terminal 68 amino acids
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26400
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SDS-PAGE
45000 - 50000
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gel filtration
120000
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sucrose density gradient centrifugation
200000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
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multisubunit RNA processing enzyme including a ring of six RNase PH subunits
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapour diffusion method, wild-type and triple mutant R68Q-R73Q-R76Q RNase PH crystallized and the structures determined by X-ray diffraction to medium resolution. Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively
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purified recombinant mthRrp41-mthRrp42 protein complex, hanging-drop vapour diffusion, 0.001 ml of protein in 25 mM Tris pH 7.5, 0.15 M NaCl, mixed with 0.001 ml of reservoir solution containing 5% 2-propanol, 20% PEG 3350, and 0.1 M succinic acid pH 7.0, X-ray diffraction structure determination and analysis at 2.65 A resolution
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hanging-drop vapor diffusion method. The 1.9 A crystal structures of the apo and the phosphate-bound forms of RNase PH from reveal a monomeric RNase PH with an alpha/beta-fold tightly associated into a hexameric ring structure in the form of a trimer of dimers
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52
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Tm-value for wild-type enzyme and mutant enzyme R127A
55
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10 min, about 50% loss of activity
65
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10 min, complete inactivation
additional information
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enzyme mutant deficient in tRNA nucleotidyltransferase and polynucleotide phosphorylase activity displays reversible cold-sensitivity, ribosome structure and function are severely affected, particularly at lower temperatures of 31C and below
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
glutathione agarose bead chromatography and His-bind resin chromatography
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recombinant enzyme
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recombinant from overproducing strain NF1815
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recombinant His-tagged mthRrp41-mthRrp42 protein complex from Escherichi coli strain E2566 by nickel affinity chromatography and ultrafiltration
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Talon column chromatography and Source Q column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli strain BL21(DE3)pLysS
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expressed in Escherichia coli strains BL21(DE3) and HB101
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from total RNA into pBluescript II KS+ for sequencing, in pAS2 for subcloning, in pBIN35S35SEmGFP for transformation of tobacco protoplasts and expression as GFP fusion protein, in p426 for generation of three isoforms with alternative ATG start codons ATG1 (Met1), ATG2 (Met6), ATG3 (Met69)
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gene rph, overexpression in strain NF1815
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genes mth682 and mth683 encode the mthRrp41-mthRrp42 protein complex, cloning and recombinant expression of the His-tagged protein complex in Escherichia coli strain E2566
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
M6L
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exclusively localised to mitochondria
R68Q-R73Q-R76Q
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triple mutant RNase PH crystallize as a dimer, compared to wild-type enzyme that crystallizes as a hexamer
R127A
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mutation has no influence on the structure
R69S
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mutation has no influence on the structure
R69S/R77S
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mutation leads to the dissociation of RNase PH hexamer into dimers without perturbing the overall monomeric structure
R74S
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mutation leads to the dissociation of RNase PH hexamer into dimers without perturbing the overall monomeric structure
R74S/R77S
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mutation leads to the dissociation of RNase PH hexamer into dimers without perturbing the overall monomeric structure
R77S
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mutation has no influence on the structure
additional information
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enzyme mutant deficient in tRNA nucleotidyltransferase and polynucleotide phosphorylase activity grows slowly at 37C, shows a dramatically reduced tRNATyrsu3+ suppressor activity, displays reversible cold-sensitivity, and performs normal tRNA synthesis, ribosome structure and function are severely affected, particularly at lower temperatures of 31C and below
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