Information on EC 3.1.26.3 - ribonuclease III and Organism(s) Escherichia coli and UniProt Accession P0A7Y0

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This record set is specific for:
Escherichia coli
UNIPROT: P0A7Y0


The taxonomic range for the selected organisms is: Escherichia coli

The enzyme appears in selected viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.1.26.3
-
RECOMMENDED NAME
GeneOntology No.
ribonuclease III
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endonucleolytic cleavage to 5'-phosphomonoester
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric diester
-
hydrolysis of phosphoric diester
-
-
hydrolysis of phosphoric ester
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
78413-14-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
; K12
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
-
structure analysis and comparison to other enzyme family members, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
double-stranded RNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
responsible for processing of dsRNA
small duplex products of 10-18 base pairs
-
?
double-stranded RNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
double-stranded RNA + H2O
?
show the reaction diagram
-
R1.1 RNA
-
-
?
ds-rRNA + H2O
mature ds-rRNA
show the reaction diagram
dsRNA + H2O
?
show the reaction diagram
-
RNase III(E38A) generates discrete-sized products from long dsRNA
-
-
?
dsRNA + H2O
mature RNA
show the reaction diagram
dsRNA + H2O
processed RNA
show the reaction diagram
hairpin RNA R1.1 + H2O
?
show the reaction diagram
-
RNase III(E38A) cleaves at the primary site and remains bound to the RNA, thereby preventing cleavage at the secondary site
-
-
?
mRNA transcripts + H2O
5'-phosphooligonucleotides
show the reaction diagram
poly(A)-poly(U) + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
-
-
-
?
pre-16S rRNA + H2O
mature 16S rRNA
show the reaction diagram
-
-
-
-
?
pre-23S rRNA + H2O
mature 23S rRNA
show the reaction diagram
-
-
-
-
?
pre-5S rRNA + H2O
mature 5S rRNA
show the reaction diagram
-
-
-
-
?
pre-mRNA + H2O
mature mRNA
show the reaction diagram
pre-rRNA + H2O
mature rRNA
show the reaction diagram
-
-
-
-
?
R1.1 RNA + H2O
2 fragment of R1.1 RNA
show the reaction diagram
-
substrate is enzymatically synthesized based on the R1.1 processing signal, which is encoded in the phage T7 genetic early region between genes 1.0 and 1.1, 1 cleavage site
-
-
?
R1.1 RNA + H2O
2 fragments of R1.1 RNA
show the reaction diagram
R1.1 RNA + H2O
?
show the reaction diagram
-
-
-
-
?
R1.1 RNA + H2O
fragments of R1.1 RNA
show the reaction diagram
R1.1 RNA derivatives + H2O
fragments of R1.1 RNA
show the reaction diagram
-
based on the R1.1 processing signal, which is encoded in the phage T7 genetic early region between genes 1.0 and 1.1, derivative R1.1[CL3B] is not cleaved and its binding to the enzyme leads to uncoupling of substrate recognition and cleavage
-
-
?
ribosomal RNA + H2O
smaller precursor rRNA
show the reaction diagram
RNA precursor + H2O
mature RNA
show the reaction diagram
-
phage lambda RNA, enzyme is involved in translation control
-
-
?
RNA substituted with guanosine 5'-O-(1-thiotriphosphate) + H2O
5'-phosphooligonucleotides containing guanosine 5'-O-(1-thiotriphosphate)
show the reaction diagram
-
cleavage specifity is not altered by modified RNA
-
-
?
single-stranded RNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
tRNA + H2O
5'-phosphooligonucleotides
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
double-stranded RNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
P0A7Y0
responsible for processing of dsRNA
small duplex products of 10-18 base pairs
-
?
double-stranded RNA + H2O
5'-phosphooligonucleotides
show the reaction diagram
-
cleaves multimeric tRNA precursor at the spacer region, also involved in processing of precursor rRNA, hnRNA and early T7-mRNA. Also cleaves double-stranded DNA and single-stranded RNA
-
-
?
ds-rRNA + H2O
mature ds-rRNA
show the reaction diagram
-
-
-
-
?
dsRNA + H2O
mature RNA
show the reaction diagram
dsRNA + H2O
processed RNA
show the reaction diagram
-
-
-
-
?
pre-16S rRNA + H2O
mature 16S rRNA
show the reaction diagram
-
-
-
-
?
pre-23S rRNA + H2O
mature 23S rRNA
show the reaction diagram
-
-
-
-
?
pre-5S rRNA + H2O
mature 5S rRNA
show the reaction diagram
-
-
-
-
?
pre-mRNA + H2O
mature mRNA
show the reaction diagram
-
enzyme regulates gene expression by controlling mRNA translation and stability
-
-
?
pre-rRNA + H2O
mature rRNA
show the reaction diagram
-
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
can substitute for Mg2+
Mg2+
; required
Mn2+
can substitute for Mg2+
Ni2+
can substitute for Mg2+
Zn2+
-
inhibits Mg2+ dependent reaction
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-hydroxyisoquinoline-1,3-dione
-
inhibits cleavage of R1.1 RNA, IC50: 0.014 mM for Mg2+-supported reaction, 0.008 mM for Mn2+-suppoeted reaction, noncompetitive inhibition
2-mercaptoethanol
-
required
Double-stranded RNA
-
inhibits cleavage of single-stranded RNA
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Ethidium bromide
F-
-
inhibits at 100 mM
inhibitory base pair sequences within a RNA substrate
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inclusion of disfavored base pair sequences inhibit activity
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KCl
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enzyme is sensitive to high salt conditions, above 50 mM, the mutant homodimer and the heterodimer are more sensitive than the wild-type
poly(IC)
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as double-stranded RNA
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tRNA
-
inhibits enzyme activity and inhibition by ethidium bromide
YmdB
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stress-responsive ribonuclease-binding regulator of Escherichia coli RNase III activity. Interacting with a site in the RNase III catalytic region. Expression of YmdB is transcriptionally activated by both cold-shock stress and the entry of cells into stationary phase, and that this activation requires the delta-factor-encoding gene, rpoS
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
-
less than 1 mM
glutamate
-
extends salt concentration range
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
38
Mn2+
-
mutant E117D, pH 7.5, 37C
0.000041 - 0.003
R1.1 RNA
-
0.00026 - 0.34
RNA
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00035 - 0.72
R1.1 RNA
-
3.8 - 7.7
RNA
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017
Ethidium bromide
-
pH 8.0, 37C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014
2-hydroxyisoquinoline-1,3-dione
Escherichia coli
-
inhibits cleavage of R1.1 RNA, IC50: 0.014 mM for Mg2+-supported reaction, 0.008 mM for Mn2+-suppoeted reaction, noncompetitive inhibition
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
gel mobility shift assay for substrate bindng determination
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 8.2
-
-
7 - 7.4
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inactive below, RNA
7.5
-
assay at
7.9
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.9 - 7.4
-
-
7.6 - 9.75
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-
8 - 10
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 37
-
-
30 - 45
-
no activity at 0C and 17.5C
additional information
-
samples of bacterial cultures are grown in LB medium at 28C or 37C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000 - 55000
41000
-
RNase O, gel filtration
54000
-
recombinant His-tagged mutant homodimer, gel filtration
78000
-
recombinant His-tagged and GST-tagged wild-type/mutant heterodimer, gel filtration
103000
-
recombinant GST-tagged wild-type homodimer, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 25600, amino acid sequence calculation, x * 29000, recombinant His-tagged enzyme, SDS-PAGE
heterodimer
-
preparation of artificial heterodimers of RNase III, which are providing new insight on the subunit and domain interactions involved in dsRNA recognition and cleavage
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
-
phosphorylation on a serine in the RNase III domain activates the enzyme, the covalent modification facilitates product release, which is the rate-limiting step in the catalytic pathway
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
12 three-dimensional structures of bacterial RNase III in various forms have been reported
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
30 min, no loss af activity
50
-
30 min, 50% loss af activity
100
-
nonspecific cleavage of RNA after heating for 1 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 25 mM Tris, pH 8, 8% (NH4)2SO4, 50% glycerol, several months, no loss of activity
-
-70C, 1.3 M NH4Cl, 2 years, 40-50% loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography. RNase III heterodimers also can be purified from the inclusion body
-
of the overexpressed protein
-
of the overexpressed protein in yeast
-
partially RNase O
-
recombinant His-tagged enzyme to over 90% purity
-
recombinant His-tagged RNase III from Escherichia coli by nickel affinity chromatography
-
recombinant HIs-tagged wild-type and mutant enzymes
-
recombinant hybrid proteins to homogeneity
-
recombinant mutant enzymes
-
recombinant tagged heterodimer and homodimers from strain BL21 to homogeneity
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of the rnd gene in Escherichia coli
-
expressed in Escherichia coli BLR(DE3) cells
-
expression of a His-tagged E117Q mutant homodimer, a GST-tagged wild-type homodimer, and a His-tagged and GST-tagged wild-type/mutant heterodimer in strain BL21
-
gene rnc, expression of wild-type and mutant enzymes in strain BL21(DE3) as His-tagged enzymes
-
gene rnc, mapping at 55 min on the bacterial chromosome, overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
gene rnc, overexpression of wild-type and mutants in strain BL21(DE3) as His-tagged proteins
-
mutant proteins expressed in Escherichia coli
-
overexpression in Saccharomyces cerevisiae
-
overexpression of mutant hybrid proteins in Escherichia coli strain HMS174
-
overexpression of the rnc gene in Escherichia coli
-
recombinant expression of His-tagged RNase III in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Q153P
the Q153P substitution in the middle of the flexible linker between the endoND and the dsRBD abolish RNA-cleavage activity
D114A
-
mutant exhibits catalytic activity in vitro in 10 mM Mg2+ buffer that is comparable to that of the wild-type enzyme. At 1 mM Mg2+, the activity is significantly lower, KM-value for Mg2+ is about 2.8fold larger than the wild-type value
D45A
-
mutant enzyme exhibits negligible activity, regardless of the Mg2+ concentration
D45E
-
activity is partially rescued by Mn2+; mutant enzyme exhibits negligible activity, regardless of the Mg2+ concentration
D45N
-
mutant enzyme exhibits negligible activity, regardless of the Mg2+ concentration
E100A
-
mutant enzyme requires higher Mg2+ concentrations for optimal activity than the wild-type enzyme
E117D
-
site-directed mutagenesis, mutant exhibits normal homodimeric behaviour, can bind substrates but shows highly reduced hydrolysis activity compared to the wild-type enzyme
E41A
-
mutant exhibits catalytic activity in vitro in 10 mM Mg2+ buffer that is comparable to that of the wild-type enzyme. At 1 mM Mg2+, the activity is significantly lower, KM-value for Mg2+ is about 2.8fold larger than the wild-type value
E41A/D114A
-
KM-value for Mg2+ is about 85fold larger than the wild-type value
E65A
-
mutant enzyme requires higher Mg2+ concentrations for optimal activity than the wild-type enzyme
G97E
-
increases requirement for Mg2+
additional information