Information on EC 3.1.27.10 - rRNA endonuclease

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

EC NUMBER
COMMENTARY hide
3.1.27.10
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RECOMMENDED NAME
GeneOntology No.
rRNA endonuclease
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of the phosphodiester linkage between guanosine and adenosine residues at one specific position in 28S rRNA from rat ribosomes
show the reaction diagram
also acts on bacterial rRNA
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
endoribonuclease reaction
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hydrolysis
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CAS REGISTRY NUMBER
COMMENTARY hide
1407-48-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
alpha-sarcin-like ribotoxin restrictocin
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
MG1363
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Manually annotated by BRENDA team
MG1363
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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mutations in alpha-sarcin, which impair alpha-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate Jun N-terminal kinase, thus the sarcin-ricin loop remains intact indicating that the alpha-sarcin mutants are catalytically inactive
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
23S rRNA + H2O
?
show the reaction diagram
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cleavage of sarcin-ricin loop of 23S rRNA inhibits in vitro translation, slightly affects binding of elongation factor Tu ternary complex to the ribosome, inhibits elongation factor G binding, and consequently GTP hydrolysis and mRNA-tRNA translocation
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?
28 S rRNA + H2O
alpha-fragment from the 3' end of 28S rRNA
show the reaction diagram
70S ribosomes + H2O
?
show the reaction diagram
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?
ApA + H2O
3'-AMP
show the reaction diagram
ApA + H2O
3'-AMP + adenosine
show the reaction diagram
CpC + H2O
?
show the reaction diagram
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?
GpA + H2O
3'-GMP
show the reaction diagram
RNA + H2O
?
show the reaction diagram
rRNA + H2O
?
show the reaction diagram
SRL + H2O
?
show the reaction diagram
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sarcin/ricin loop in ribosomal RNA
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?
supercoiled double-stranded DNA + H2O
nicked circular conformation of DNA
show the reaction diagram
yeast 25 S RNA + H2O
?
show the reaction diagram
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?
yeast 26 S RNA + H2O
?
show the reaction diagram
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alpha-sarcin cleaves the phosphodiester bond between the guanine residue at position 3025 and the adenine residue at position 3026
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?
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
rRNA + H2O
?
show the reaction diagram
additional information
?
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the ribosome-inactivating proteins alpha-sarcin targets the ribosomal sarcin/ricin loop, specific recognition and cleavage. The eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the sarcin/ricin loop, overview. alpha-Sarcin does not interact with the P1/P2 C-terminal peptide SDDDMGFGLFD
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
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at low salt concentrations less than 5 mM maximum of cleavage rate; at low salt concentrations of less than 5 mM the enzyme has its maximum cleavage rate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cibacron blue F3GA
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Mg2+
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inhibits hydrolysis of RNA
Sodium dodecyl sulfate
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reversible inhibition of ribonuclease activity, irreversible inhibition of deoxyribonuclease activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Mg2+
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strong decrease of efficiency of oligonucleotide hybridization
ribosomal stalk
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the eukaryotic ribosomal stalk directly interacts with several members of the N-glycosidase family, favoring their disruption of the SRL
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additional information
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P1 protein isozymes P1alphaP2beta and P1betaP2alpha are acidic proteins that are extremely dynamic, including their exchange with the cytoplasmic pool, their C-terminal regions being responsible for the interaction, recruitment, and regulation of supernatant translation factors and ribosome-inactivating proteins like ricin and trichosanthin, but alpha-sarcin does not interact with the P1/P2 C-terminal peptide SDDDMGFGLFD
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.027 - 0.045
ApA
0.0036
GpA
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pH 7.0, 25°C
0.076
SRL
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sarcin/ricin loop in ribosomal RNA, 10 mM Tris-HCl, 0-5 mM KCl, pH 7-7.4, 37°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.917
28 S rRNA
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0.000012 - 0.00027
ApA
0.000051
CpC
Aspergillus giganteus
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0.000009
GpA
Aspergillus giganteus
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pH 7.0, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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specific activity 18200 units/mg, 1 unit is the amount of protein causing 50% inhibition of protein synthesis in 0.001 ml of rabbit-reticulocyte lysate system
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.5 - 8.5
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no activity below pH 2.5 or above pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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both mutant and wild-type variants of alpha-sarcin localize to the nucleus and cytoplasm, where they colocalize with ribosomal marker RPS6
Manually annotated by BRENDA team
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both mutant and wild-type variants of alpha-sarcin localize to the nucleus and cytoplasm, where they colocalize with ribosomal marker RPS6
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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x * 45000, recombinant His-tagged enzyme fusion construct scFvA33alphasarcin, SDS-PAGE
additional information
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15N-1H residual dipolar coupling NMR, alkyl poly(ethylene glycol)/alcohol mixture, 1 mM, pH 6.0, 25°C
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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dimeric form fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, but is an effective ribonuclease in situ
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ribonucleoprotein
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
15N-1H residual dipolar coupling NMR, alkyl poly(ethylene glycol)/alcohol mixture, 1 mM, pH 6.0, 25°C, 24 Hz
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alpha-sarcin three-dimensional structure determined by X-ray diffraction of single crystals of the toxin
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 8.5
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
54
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L145F mutant variant, increased transition temperature Tm
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
23SrRNA isolated from Escherichia coli 50S ribosomal subunits
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C-25 SP ion-exchange column, pH 5.5, 4°C
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deletion mutant DELTA(7-22)
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L145F mutant variant
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to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alpha-sarcin cDNA cloned by consecutive annealing of overlapping primers followed by ligation into XhoI- and HindIII-sites in pcDNA3.1 or pcDNA3-mychis vector (C-terminal MycHis tag). alpha-Sarcin cDNA cloned into 3 × FLAG pMSCV to obtain a N-terminal 3 × Flag tagged version of alpha-sarcin. Expression of wild-type and mutant constructs in HeLa or Cos-7 cells
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cloned and expressed in Escherichia coli
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cloned and expressed in Escherichia coli BL21(DE3)
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cloned and expressed in Escherichia coli RB791
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cloned and expressed in Escherichia coli TG2
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cloning of the genomic alpha-sarcin gene pGEM-T/FL-alpha plasmid, construction of the alpha-sarcin gene and its mutants by PCR, using genomic DNA as template
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deletion mutant delta(7-22) produced in Escherichia coli
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design of RNA fragment ECAS171
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doxycycline-inducible intracellular expression of wild-type alpha-sarcin in Saccharomyces cerevisiae strain W303 and in strains deficient for P1 or P2 protein, wild-type alpha-sarcin is lethal in all wild-type yeast clones, while 30% and 80% of resistant clones are obtained for the DELTAP1alphaP2beta and DELTAP2 strains, respectively
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mutants K63D, R21D/K28D and R21D/K28D/K63D
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recombinant expression of FLAG3-tagged alpha-sarcin in HeLa cells, alpha-sarcin splicing is incduced by and dependent on rapamycin, is inducible with rapid kinetics, and triggers apoptosis in the cytoplasm of HeLa cells
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recombinant extracellular His-tagged enzyme fusion construct scFvA33alphasarcin from expression in Pichia pastoris strain KM71 by nickel affinity chromatgraphy; the His-tagged enzyme is fused to the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33, the construct scFvA33alphasarcin is expressed in Pichia pastoris strain KM71 using the AOX1 promoter, subcloning in Escherichia coli DH5aF cells, and secretion to the medium
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C148S
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point mutation constructed from cDNA clone
C6S
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point mutation constructed from cDNA clone
E125I
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site-directed mutagenesis
E96A
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mutagenised using the T7-GEN TM in vitro mutagenesis kit, 80% less cytotoxity to HeLa cells
E96Q
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mutagenesis variant protein
H50/137Q
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mutagenesis variant protein
H50/137Q/E96Q
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mutagenesis variant protein
H50Q
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mutagenesis variant protein
H50Q/H137Q/E96Q
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catalytically inactive alpha-sarcin mutant sar3M
I129C
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site-directed mutagenesis
L145F
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oligonucleotide-site directed mutagenesis, retains cytotoxic activity
R121K
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oligonucleotide site-directed mutagenesis
Y48F
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mutant is produced in Escherichia coli and purified to homogeneity (ca. 3 mg/l of original bacterial culture)
E96A
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mutagenised using the T7-GEN TM in vitro mutagenesis kit, 80% less cytotoxity to HeLa cells
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E96Q
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mutagenesis variant protein
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H137Q
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mutagenesis variant protein; substitution abolishes the catalytic activity
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H50/137Q
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mutagenesis variant protein
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H50/137Q/E96Q
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mutagenesis variant protein
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H50Q
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mutagenesis variant protein
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K63D
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mutant with decreased activity for sarcin/ricin loop-cleavage of ribosomes
R21D/K28D
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mutant with decreased activity for sarcin/ricin loop-cleavage of ribosomes
R21D/K28D/K63D
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mutant with decreased activity for sarcin/ricin loop-cleavage of ribosomes
K11E
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slightly lower specific ribonucleolytic activity than wild type (assayed against intact ribosomes)
K14E
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also able to cleave poly(C)
K17E
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slightly lower specific ribonucleolytic activity than wild type (assayed against intact ribosomes)
K21E
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slightly lower specific ribonucleolytic activity than wild type (assayed against intact ribosomes)
R22E
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slightly lower specific ribonucleolytic activity than wild type (assayed against intact ribosomes)
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine