Information on EC 2.7.7.52 - RNA uridylyltransferase

Word Map on EC 2.7.7.52
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.7.52
-
RECOMMENDED NAME
GeneOntology No.
RNA uridylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UTP + RNAn = diphosphate + RNAn+1
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
UTP:RNA uridylyltransferase
The enzyme requires an oligoribonucleotide or polyribonucleotide with a free terminal 3'-OH as a primer.
CAS REGISTRY NUMBER
COMMENTARY hide
78519-53-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UC strain
-
-
Manually annotated by BRENDA team
isoform Rsp1
UniProt
Manually annotated by BRENDA team
strain TREU-667
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + (mRNA)n
diphosphate + (mRNA)n+1
show the reaction diagram
-
i.e. total human mRNA
-
-
?
ATP + 5'(ppp)-UGAGGUAGUAGGUUGUAUAGUU-3'
diphosphate + 5'(ppp)-UGAGGUAGUAGGUUGUAUAGUUA-(p)-3'
show the reaction diagram
-
i.e. triphosphorylated human let-7a-5p-3p
-
-
?
ATP + 5'-UGAGGUAGUAGGUUGUAUAGUU-3'
diphosphate + 5'-UGAGGUAGUAGGUUGUAUAGUUA-(p)-3'
show the reaction diagram
-
i.e. unphosphorylated human let-7a-0P. Gld2 displays an 83fold preference of ATP over UTP
-
-
?
ATP + RNAn
diphosphate + RNAn+1
show the reaction diagram
CTP + 5'(pp)-UGAGGUAGUAGGUUGUAUAGUU-3'
diphosphate + 5'(pp)-UGAGGUAGUAGGUUGUAUAGUUC-(p)-3'
show the reaction diagram
-
i.e. diphosphorylated human let-7a-5p-2p
-
-
?
CTP + RNAn
diphosphate + RNAn+1
show the reaction diagram
GTP + miR-122
diphosphate + ?
show the reaction diagram
-
-
-
-
?
GTP + RNAn
diphosphate + RNAn+1
show the reaction diagram
UTP + 5'(p)-UGAGGUAGUAGGUUGUAUAGUU-3'
diphosphate + 5'(p)-UGAGGUAGUAGGUUGUAUAGUUU-(p)-3'
show the reaction diagram
-
i.e. monophosphorylated human let-7a-5p. Gld2 displays an 83fold preference of ATP over UTP
-
-
?
UTP + RNAn
diphosphate + RNAn+1
show the reaction diagram
UTP + RNAn containing a terminal G residue
diphosphate + RNAn+1
show the reaction diagram
-
substrate dsRNA
-
-
?
UTP + RNAn containing a terminal U residue
diphosphate + RNAn+1
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
UTP + RNAn
diphosphate + RNAn+1
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
influences the TUT4 conformation
KCl
-
stimulated by concentrations up to 50 mM
additional information
divalent cations essential for catalysis
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aurintricarboxylic acid
-
-
brRNA
-
inhibits the number of added residues
-
Cibacron blue F3GA
-
-
diphosphate
-
-
heparin
Ionic strength
-
quite sensitive to ionic strength, activity decreases by 50% at about 40 mM (NH4)2SO4 or 125 mM potassium acetate
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ionic strength
-
stimulates, e.g. 600 mM KCl
-
protein TbMP81
-
editosome protein, interaction enhances the enzyme activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000228
ATP
-
pH 8.0, 37°C
0.055
CTP
-
pH 8.0, 37°C
0.23
GTP
-
pH 8.0, 37°C
0.0002 - 0.132
RNA
0.0004 - 0.0025
RNAn
0.0126 - 0.0208
RNAn containing a terminal U residue
-
0.0004 - 48
UTP
additional information
additional information
steady-state enzyme kinetics, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000293
ATP
Homo sapiens
-
pH 8.0, 37°C
0.000914
CTP
Homo sapiens
-
pH 8.0, 37°C
0.000403
GTP
Homo sapiens
-
pH 8.0, 37°C
0.00166 - 0.0216
RNA
0.0003 - 0.0007
RNAn
0.0015 - 0.0033
RNAn containing a terminal U residue
-
0.00002 - 0.021
UTP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.128
ATP
Homo sapiens
-
pH 8.0, 37°C
4
0.0166
CTP
Homo sapiens
-
pH 8.0, 37°C
60
0.0018
GTP
Homo sapiens
-
pH 8.0, 37°C
37
0.133 - 0.167
RNAn
2667
0.067 - 0.267
RNAn containing a terminal U residue
19746
0.0015 - 0.6
UTP
65
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.67
-
purified enzyme
8.2
-
purified recombinant enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
-
assay at
8.2
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37800
gel filtration
43000
-
gel filtration
50500
-
velocity sedimentation
68000
-
gel filtration
115000
-
x * 115000, U6-TUTase, SDS-PAGE
135000
-
native PAGE
140000
-
4 * 140000, TUT1
500000
700000
-
RNA-stabilized higher complexed form, native PAGE and glycerol gradient sedimentation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
-
-
tetramer
-
4 * 140000, TUT1
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant N-terminally His-tagged TUT4, free or with bound 2'-deoxyribonucleoside, at a concentration of 10 mg/mL in 10 mM HEPES, pH 7.6, 70 mM KCl, 0.5 mM DTT, and in presence of 4 mM MgCl2 and 0.05 mM UTP, screening with commercial crystallization screens, vapor diffusion technique, 4°C, cryoprotection by 15% glycerol, X-ray diffraction structure determination and analysis at 2.0-2.4 A resolution; small plate-like co-crystals of purified recombinant N-terminally His-tagged TbTUT4 and UTP grow in 100 mM sodium cacodylate (pH 6.5), 200 mM calcium acetate, 18% (w/v) PEG-8000, crystal strucuture reveals two significantly different conformations of this TUTase:one molecule is in a relatively open apo confirmation, whereas the other displays a more compact TUTase-UTP complex
structure of apoenzyme and in complex with UTP, to 1.56 A and 1.95 A resolution, respectively. Enzyme possesses a bridging domain, which extends from the C-terminal domain and makes hydrophobic contacts with the N-terminal domain, thereby creating a cavity adjacent to the UTP-binding site. Enzyme shows no appreciable conformational change upon UTP binding and apparently does not require RNA substrate to select a cognate nucleoside triphosphate.
TUT4 in complex with UTP, X-ray diffraction structure analysis
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
at least 3 h
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
protein is stable at -20°C for several months
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, 25% glycerol, stable
-
4°C, does not tolerate more than several h at
-
protein remains stable at -20°C in 50% glycerol up to 3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2400fold to near homogeneity
-
614fold from uninfected and 117fold from CPMV-infected leaves
-
expression and purification of 20S editosome-associated RET2 complex from Trypanosoma brucei by affinity isolation; recombinant RET1 from Trypanosoma brucei is purified by metal affinity chromatography (Talon metal affinity column), anion-exchange chromatography (HiTrap Q column) and loading onto a MonoS 5/5 column (final purification step), expected yield is 0.5 mg/liter of bacterial culture, it is important to maintain the concentration of KCl above 80 mM during purification, as protein tends to precipitate under low salt conditions; the recombinant expressed RET2 is purified by loading the protein extract onto a Talon affinity column and onto a HiTrap Sepharose S column, the expected protein yield is 1 mg of protein per liter of bacterial culture
-
homogenous RET1 is purified from an enriched mitochondrial fraction of Leishmania tarentolae by a series of chromatographic steps: affinity chromatography (polyU Sepharose 4B), anion-exchange chromatography (Poros 20 HQ 46/100 column), gel filtration (Superose 12 and Superose 6 columns) and hydrophobic interactions chromatography (Phenyl Superose column); recombinant RET1 from Leishmania tarentolae is purified by cation-exchange resin (Sepharose S Fast Flow), metal affinity chromatography (Talon metal affinity column), anion-exchange chromatography (Mono Q 5/5 column), expected yield: 0.2-0.5 mg/liter
-
partial purification of U6 snRNA specific enzyme form via chromatography on a U6 snRNA affinity resin, separation from unspecific enzyme form
-
recombinant enzyme
-
recombinant His-tagged enzyme from Escherichia coli
-
recombinant His-tagged wild-type and mutant TUT4 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ion exchange chromatography, and gel filtration
recombinant TAP-tagged enzyme from Leishmania tarentolae cells
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in Trypanosoma brucei cells strain 29.13
-
DNA sequence determination, editosomal enzyme, expression as His-tagged protein in Escherichia coli
-
DNA sequence determination, gene is located on chromosome 9, expression as His-tagged protein in Escherichia coli
-
DNA sequence determination, mitochondrial enzyme, expression as His-tagged protein in Escherichia coli
-
expressed in Escherichia coli BL-21 as a 6xHis tagged fusion protein; TUT4, DNA and amino acid sequence determination and analysis, expression of N-terminally His-tagged wild-type and mutant TUT4 in Escherichia coli strain BL21(DE3)
expression in Escherichia coli BL21(DE3) Codon Plus RIL, use of strains bearing additional tRNAs for expression of genes with G-rich codons is essential for RET1 expression
-
expression in Escherichia coli BL21(DE3) Codon Plus RIL, use of strains bearing additional tRNAs for expression of genes with G-rich codons is essential for RET1 expression; for recombinant expression in Escherichia coli BL21 the RET2 gene lacking 21 codons at the 5' end, which correspond to the mitochondria importation signal, is cloned into pET15+vector to generate N-terminal 6 His-RET2 fusion protein; TbRET2 fused with the affinity TAP tag at the C-terminus using pLew79 vector in 29-13 cell line
-
expression in HEK-293 cell; expression in HEK-293 cell
-
expression of RET2 in Leishmania tarentolae cells as cytosolic enzyme by removing the mitochondrial targeting signal sequence
-
phylogenetic analysis, sequence comparison and signature motifs, overview
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D491A/D493A
C83F
-
230% of wild-type activity for transfer to terminal U residue
D297A
Km=0.1728 (UTP), kcat=0.00033/sec (UTP)
D297N
Km=0.0139 (UTP), kcat=0.0000666/sec (UTP)
D97A
-
complete inactivation of uridylyl transfer
E300A
Km=0.3084 (UTP), kcat=0.0066/sec (UTP)
K149A
-
mutant retains partial activity
N147A
Km=0.1153 (UTP), kcat=0.005/sec (UTP)
R121A
Km=0.0028 (UTP), kcat=0.000033/sec (UTP),Km=0.132 (RNA), kcat= 0.005/sec (RNA)
R144A
-
almost complete inactivation of uridylyl transfer
R307A
Km=0.0004 (UTP), kcat=0.000133/sec (UTP), Km=0.01 (RNA), kcat= 0.00833/sec (RNA)
R435A
-
almost complete inactivation of uridylyl transfer
S148A
Km=0.0873 (UTP), kcat=0.021/sec (UTP), Km=0.0002 (RNA), kcat= 0.0166/sec (RNA)
S188A
Km=0.034 (UTP), kcat=0.0005/sec (UTP), Km=0.0003 (RNA), kcat= 0.00166/sec (RNA)
V271R
-
10-20% of wild-type activity
V271R/C83F
-
240% of wild-type activity for transfer to terminal U residue
Y189F
Km=0.0213 (UTP), kcat=0.015/sec (UTP)
Y319A
-
complete inactivation of uridylyl transfer
additional information
-
mutations of metal coordinating catalytic aspartic residues D66, D68 and D136, render TUT4 inactive but have no effect on UTP binding
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
a single C2H2-type zinc finger domain of Zcchc11 is responsible for the functional interaction with pluripotency factor Lin28. Lin28 uses terminal uridylyltransferases Zcchc11 and Zcchc6 to control let-7 expression and has important implications for stem cell biology as well as cancer; pluripotency factor Lin28 uses terminal uridylyltransferases Zcchc11 and Zcchc6 to control let-7 expression and has important implications for stem cell biology as well as cancer