Information on EC 3.1.13.1 - exoribonuclease II

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

EC NUMBER
COMMENTARY
3.1.13.1
-
RECOMMENDED NAME
GeneOntology No.
exoribonuclease II
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
can act as a endonuclease too
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
acts in a processive manner
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
acts in a processive manner
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
can act as a endonuclease too
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
acts in a processive manner
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
acts in a processive manner
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
sensitive to RNA secondary structures
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
acts in a processive manner
-
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates
show the reaction diagram
exonucleolytic cleavage in the 3'-5'-direction to yield 5'-phosphomononucleotides
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis
-
-
hydrolysis
-, Q88DE8
-
hydrolysis
Escherichia coli JM109
-
-
-
hydrolysis of phosphoric ester
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
tRNA processing
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3'-5'exoribonuclease
-
-
3'-5'exoribonuclease
-
-
3'-5'exoribonuclease
Trypanosoma brucei 427
-
-
-
3-5exoribonuclease
-
-
Dis3
-
-
ribonuclease 2
-
-
ribonuclease II
-
-
-
-
ribonuclease II
-
-
ribonuclease II
P30850
-
ribonuclease II
Escherichia coli JM109
-
-
-
ribonuclease II
-
-
ribonuclease Q
-
-
-
-
Ribonuclease R
Q88DE8
-
RNase 2
-
-
RNase A
Saccharomyces cerevisiae YJJ
-
-
-
RNase II
-
-
RNase II
P30850
-
RNase II
Escherichia coli JM109, Escherichia coli JM83, Escherichia coli SK4803
-
-
-
RNase II
-
-
RNase II
-
-
RNase R
Escherichia coli JM83
-
-
-
RNase R
Legionella pneumophila JR32
-
-
-
RNase R
Q88DE8
-
RNase-2
-
-
Rrp44
-
-
exonuclease ISG20
-
-
additional information
-
RNase II and RNase R are the two Escherichia coli exoribonucleases that belong to the RNase II superfamily of enzymes
CAS REGISTRY NUMBER
COMMENTARY
37288-24-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
nematode, CB4856, ERI-1 mutants (mg366)
-
-
Manually annotated by BRENDA team
Chlamydomonas sp.
-
-
-
Manually annotated by BRENDA team
strain JM109 and BL21(DE3)
-
-
Manually annotated by BRENDA team
strain JM83
-
-
Manually annotated by BRENDA team
strain SK4803
-
-
Manually annotated by BRENDA team
Escherichia coli JM109
strain JM109 and BL21(DE3)
-
-
Manually annotated by BRENDA team
Escherichia coli JM83
strain JM83
-
-
Manually annotated by BRENDA team
Escherichia coli SK4803
strain SK4803
-
-
Manually annotated by BRENDA team
human ERI-1, orthologue to Caenorhabditis elegans ERI-1
-
-
Manually annotated by BRENDA team
strain JR32
-
-
Manually annotated by BRENDA team
Legionella pneumophila JR32
strain JR32
-
-
Manually annotated by BRENDA team
mouse
-
-
Manually annotated by BRENDA team
no activity in archaebacteria
except Halobacterium species NRC-1
-
-
Manually annotated by BRENDA team
no activity in Escherichia coli
strain SK4803, carrying the mutant allele rnb296, single substitution of aspartate 209 for asparagine
-
-
Manually annotated by BRENDA team
no activity in Escherichia coli SK4803
strain SK4803, carrying the mutant allele rnb296, single substitution of aspartate 209 for asparagine
-
-
Manually annotated by BRENDA team
strain KT2440
UniProt
Manually annotated by BRENDA team
mitochondrial precursor
UniProt
Manually annotated by BRENDA team
starin BJ5464
-
-
Manually annotated by BRENDA team
Saccharomyces cerevisiae BJ5464
starin BJ5464
-
-
Manually annotated by BRENDA team
Saccharomyces cerevisiae YJJ
strain YJJ
-
-
Manually annotated by BRENDA team
strains T4 and TIGR4
Uniprot
Manually annotated by BRENDA team
cyanobacteria
-
-
Manually annotated by BRENDA team
strain 427 and 29-13
-
-
Manually annotated by BRENDA team
Trypanosoma brucei 427
strain 427 and 29-13
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
plays an important role in RelE-independent A-site cleavage
malfunction
-
instead of A-site cleavage, translational pausing in DELTARNase II cells produces transcripts that are truncated +12 and +28 nucleotides downstream of the A-site codon. Deletion of RNase R has little effect on A-site cleavage. Polynucleotide phosphorylase overexpression restores A-site cleavage activity to DELTARNase II cells
additional information
-
stability of RNAse II-RNA interactions and effects on the enzyme reaction mechanism processing and degrading RNA molecules, analysis by surface plasmon resonance and electrophoretic mobility shift Assay, overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5' end-labeled pre-tRNASer + H2O
?
show the reaction diagram
P39112, -
degradation of pre-tRNASer in a processive manner, leaving a short oligonucleotide (about 3nt) product
-
-
?
A(17) + H2O
AMP + ?
show the reaction diagram
-
full-length RNase R has similar activity on both poly(A) and A(17) substrates. Full-length RNase II is 20fold more active on A(17) than full-length RNase R
-
-
?
A(4) + H2O
AMP + ?
show the reaction diagram
-
poor substrate, is degraded by RNase R 400fold more slowly than A(17)
-
-
?
dsRNA + H2O
?
show the reaction diagram
-
-
-
-
?
dsRNA + H2O
?
show the reaction diagram
-
-
-
-
?
dsRNA + H2O
?
show the reaction diagram
Q08162, -
Rrp44 is very efficient in degrading a duplex with a 30 overhang of 14 nucleotides and is inactive with overhangs as short as 2 or 3 nucleotides
-
-
-
duplex RNA + H2O
?
show the reaction diagram
-
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
mRNA-degradation
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
mRNA-maturation
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
Escherichia coli, Escherichia coli SK4803
-
synthetic mRNA substrates, either with or without a poly(A) tail, turn out to be a good choice for mimicking the actual in vivo enzyme substrates
-
-
-
mRNA + H2O
?
show the reaction diagram
-
purified RNase II is unable to directly catalyse A-site cleavage in vitro, RNase II-catalysed degradation of mRNA to the ribosome border is a prerequisite for A-site cleavage. Degrades ribosome-bound mRNA to positions +18 nucleotides downstream of the ribosomal A site
-
-
?
oligonucleotide + H2O
?
show the reaction diagram
-
-
-
-
?
oligoribonucleotide + H2O
?
show the reaction diagram
-
final end product of RNase II is 4-nt, whereas for RNase R it is a 2-nt fragment
-
-
?
oligoribonucleotide + H2O
?
show the reaction diagram
-
final end product of Rrp44 is 4-nt
-
-
?
poly (A)
5'-AMP
show the reaction diagram
Saccharomyces cerevisiae, Saccharomyces cerevisiae BJ5464
-
-
-
-
?
poly(A)
5'-AMP
show the reaction diagram
-
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
35-nucleotide poly(A) chain, RNase II final end product contains four nucleotides, while RNase R renders a two-nucleotide fragment as the final product
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
C6GKN7
35-nucleotide poly(A) chain, RNase R renders a two-nucleotide fragment as the final product
-
-
?
poly(A) + H2O
5'-AMP + oligonucleotide
show the reaction diagram
-
35-nucleotide poly(A) chain, Tyr-313 and Glu-390 are crucial for RNA specificity of RNase II, Arg-500 residue in the active site is crucial for activity but not for RNA binding. Inside the cavity the unique specific contacts for ribose established by RNase II are those with the 2nd and 4th nucleotides from the 3'-end of the RNA molecule. These contacts are necessary and sufficient for cleavage to occur, and therefore, they seem to be responsible for the RNA specificity versus DNA in RNase II
-
-
?
poly(A) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(A) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(A) + H2O
5'-AMP + oligo(A)
show the reaction diagram
-
full-length RNase R has similar activity on both poly(A) and A(17) substrates
-
-
?
poly(A) + H2O
5'-AMP + oligo(A)
show the reaction diagram
Escherichia coli, Escherichia coli SK4803
-
RNase II has a strong preference for poly(A) stretches and is highly efficient in degrading poly(A) tails. RNase II is responsible for 90% of the exonucleolytic degradation of synthetic RNA poly(A) homopolymers
-
-
?
poly(C)
5'-CMP
show the reaction diagram
-
-
-
-
?
poly(C) + H2O
5'-CMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(C) + H2O
5'-CMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(C) + H2O
5'-CMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(G)
5'-GMP
show the reaction diagram
-
-
-
-
?
poly(U)
5'-UMP
show the reaction diagram
-
-
-
-
?
poly(U) + H2O
5'-UMP + oligonucleotide
show the reaction diagram
-
-
-
?
poly(U) + H2O
5'-UMP + oligonucleotide
show the reaction diagram
-
-
-
?
polyadenosine
?
show the reaction diagram
-
23 units oligonucleotide
-
-
?
poly[8-3H]adenylic acid + H2O
?
show the reaction diagram
Escherichia coli, Escherichia coli JM109
-
linear substrate, activity below 325 UEmicro g-1
-
-
?
precursor tRNA + H2O
mature tRNA + 5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
precursor tRNA + H2O
mature tRNA + 5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
RNA
5'-phosphomononucleotides
show the reaction diagram
-
3' to 5'direction only
-
-
?
RNA
5'-phosphomononucleotides
show the reaction diagram
-
synthesized using T3 RNA polimerase
-
-
?
RNA
5'-phosphomononucleotides
show the reaction diagram
Saccharomyces cerevisiae, Saccharomyces cerevisiae BJ5464
-
Tyrosine aminotransferase RNA with 5'-phosphate terminus
-
-
?
RNA
nucleoside 5'-monophosphate
show the reaction diagram
-
-
-
-
?
RNA + H2O
phosphomononucleotides
show the reaction diagram
-, Q88DE8
RNA turnover
-
-
?
rRNA
5'-phosphomononucleotides
show the reaction diagram
Saccharomyces cerevisiae, Saccharomyces cerevisiae BJ5464
-
-
-
-
?
rRNA + H2O
?
show the reaction diagram
-
-
-
-
?
rRNA + H2O
?
show the reaction diagram
Escherichia coli, Escherichia coli JM83
-
RNase R
-
-
?
rRNA + H2O
?
show the reaction diagram
Saccharomyces cerevisiae YJJ
-
-
-
-
?
siRNA + H2O
?
show the reaction diagram
-
with 2-nt 3' overhangs
-
-
?
siRNA + H2O
?
show the reaction diagram
-
with 2-nt 3 overhangs
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA oligonucleotides with chain lengths less than seven + H2O
5'-phosphomononucleotides
show the reaction diagram
-
at high concentrations
-
?
ssRNA + H2O
?
show the reaction diagram
-
-
-
-
?
ssRNA + H2O
?
show the reaction diagram
Q08162, -
50fold higher affinity for ssRNA than for a corresponding ssDNA oligonucleotide
-
-
-
ssRNA + H2O
?
show the reaction diagram
Escherichia coli SK4803
-
-
-
-
?
T4 mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
tRNA
?
show the reaction diagram
-
tRNA from Escherichia coli
-
-
?
tRNA + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
tRNA + H2O
?
show the reaction diagram
-
tRNA-maturation
-
-
?
tRNA + H2O
?
show the reaction diagram
-
RNase BN
-
-
?
tRNAiMet + H2O
?
show the reaction diagram
P39112, -
complete degradation of the hypomodified tRNA requires both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 fails to degrade tRNAi Met, showing this to be a specific Rrp44 substrate
-
-
?
m-RNA + H2O
?
show the reaction diagram
Escherichia coli, Escherichia coli JM109
-
SL9A, containing one stem-loop structure, and malE-malF operon, containing two stem-loop structures
-
-
-
additional information
?
-
-
ds RNA is not hydrolyzed
-
-
-
additional information
?
-
-
capped mRNA is not hydrolyzed
-
-
-
additional information
?
-
-
ds DNA is not hydrolyzed
-
-
-
additional information
?
-
-
3'-phosphorylated RNA is not hydrolyzed
-
-
-
additional information
?
-
-
3'-phosphorylated RNA is not hydrolyzed
-
-
-
additional information
?
-
-
3'-phosphorylated RNA is not hydrolyzed
-
-
-
additional information
?
-
-
poly(G) is not hydrolyzed
-
-
-
additional information
?
-
-
stem-loop structured RNA is not hydrolyzed
-
-
-
additional information
?
-
-
DNA oligomers with stem-loop structures are not hydrolyzed
-
-
-
additional information
?
-
-
helical RNA is not hydrolyzed
-
-
-
additional information
?
-
-
Dis3 is responsible for exosome core activity
-
-
-
additional information
?
-
P37202
RNase activity of Dis3 is required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Dis3 is suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region
-
-
-
additional information
?
-
-
the enzyme is involed in processing of polycistronic tRNA transcripts. Polynucleotide phosphorylase (PNPase) and RNase II are required for the removal of the 3 Rho-dependent terminator sequences
-
-
-
additional information
?
-
-, Q88DE8
mRNA is a direct substrate for RNase R
-
-
-
additional information
?
-
-, Q88DE8
RNase R is a 3'-5'-exoribonuclease that is very processive and can efficiently digest RNAs having extensive secondary structures, such as rRNA, RNAs containing repetitive extragenic palindromic (REP) sequences, or the transfer-messenger RNA required for trans-translation.
-
-
-
additional information
?
-
-, Q88DE8
RNase R is also involved in the processing of 16S and 5S rRNA
-
-
-
additional information
?
-
-, Q88DE8
The absence of RNase R leads to moderate increases in the mRNA levels of some RNases and RNA helicases, but other RNases and RNA helicases are not affected.
-
-
-
additional information
?
-
-
at optimal (37C) or elevated (42C) growth temperatures, the loss of RNase R in the RNase R mutant has no major consequence on bacterial growth and has a moderate impact on normal gene regulation. At lower temperatures (25C or 30C), the loss of RNase R has a significant impact on bacterial growth and results in the accumulation of structured RNA degradation products. Concurrently, gene regulation is affected and specifically results in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R is sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant is independent of previously identified competence regulators. The rnr mutant is competent for genetic transformation. RNase R is dispensable for the intracellular multiplication of Legionella pneumophila in both human and protozoan hosts. A physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability
-
-
-
additional information
?
-
-
RNase II is one of the major enzymes involved in mRNA processing. If the CSD is limiting the action of RNase II in vivo, it may play an important role working as a brake and thus preventing the massive degradation of RNA
-
-
-
additional information
?
-
-
cold-shock domains of RNase R appear to play a role in substrate recruitment, whereas the S1 domain is most likely required to position substrates for efficient catalysis. The nuclease domain alone, devoid of the cold-shock and S1 domains, is sufficient for RNase R to bind and degrade structured RNAs. RNase R binds RNA more tightly than the nuclease domain of RNase II
-
-
-
additional information
?
-
-
does not catalyze degradation of double-stranded RNA. RNase II has a CSD domain at the N-terminal end of the protein, a central RNB catalytic domain, and an S1 RNA-binding domain at the C-terminus. S1 domain is highly important for RNase II activity and its contribution to productive RNA binding is much more important than that of the CSD domain. This domain somehow prevents the rapid degradation of RNA by RNase II, which may be highly important to overall mRNA decay in Escherichia coli
-
-
-
additional information
?
-
-
except for the more loosely associated Rtf1, the remaining components Paf1, Ctr9, Cdc73, and Leo1 of the Paf1 complex stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin
-
-
-
additional information
?
-
-
RNase activity of PNPase is critical for its cold shock function, while its polymerization activity is dispensable. In vivo counterpart of PNPase that can compensate for its absence at low temperature reveals only one protein, another 3'-to-5' exonuclease, RNase II. RNase R, which is cold inducible, cannot complement the cold shock function of PNPase
-
-
-
additional information
?
-
-
Tyr253 and Phe358 are not essential for catalysis by RNase II. Tyr253 seems to be a critical residue in setting the smallest product generated by RNase II, thus being important for the stabilization of the 3'-end of the RNA molecule. Tyr253 is highly conserved and equivalent residues are present in many RNase II family members. Phe358 can be preventing a faster degradation of the RNA by stalling its translocation, probably due to the stacking of its aromatic ring between the bases of contiguous nucleotides. Asp201, Asp207, Asp209, and Asp210 are located in the RNase II active site. These residues are not equivalent and their functions in RNA metabolism are distinct, whereby Asp209 is the only residue essential for RNase II activity
-
-
-
additional information
?
-
-
RNase II is only able to cleave DNA bases when having a ribose in the 2nd or the 4th positions
-
-
-
additional information
?
-
-
RNase II degrades RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. RNase II activity is impaired by double-stranded RNAs
-
-
-
additional information
?
-
Escherichia coli JM83
-
RNase activity of PNPase is critical for its cold shock function, while its polymerization activity is dispensable. In vivo counterpart of PNPase that can compensate for its absence at low temperature reveals only one protein, another 3'-to-5' exonuclease, RNase II. RNase R, which is cold inducible, cannot complement the cold shock function of PNPase
-
-
-
additional information
?
-
Legionella pneumophila JR32
-
at optimal (37C) or elevated (42C) growth temperatures, the loss of RNase R in the RNase R mutant has no major consequence on bacterial growth and has a moderate impact on normal gene regulation. At lower temperatures (25C or 30C), the loss of RNase R has a significant impact on bacterial growth and results in the accumulation of structured RNA degradation products. Concurrently, gene regulation is affected and specifically results in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R is sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant is independent of previously identified competence regulators. The rnr mutant is competent for genetic transformation. RNase R is dispensable for the intracellular multiplication of Legionella pneumophila in both human and protozoan hosts. A physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability
-
-
-
additional information
?
-
Escherichia coli SK4803
-
RNase II is one of the major enzymes involved in mRNA processing. If the CSD is limiting the action of RNase II in vivo, it may play an important role working as a brake and thus preventing the massive degradation of RNA, does not catalyze degradation of double-stranded RNA. RNase II has a CSD domain at the N-terminal end of the protein, a central RNB catalytic domain, and an S1 RNA-binding domain at the C-terminus. S1 domain is highly important for RNase II activity and its contribution to productive RNA binding is much more important than that of the CSD domain. This domain somehow prevents the rapid degradation of RNA by RNase II, which may be highly important to overall mRNA decay in Escherichia coli
-
-
-
additional information
?
-
Saccharomyces cerevisiae YJJ
-
except for the more loosely associated Rtf1, the remaining components Paf1, Ctr9, Cdc73, and Leo1 of the Paf1 complex stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
mRNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
precursor tRNA + H2O
mature tRNA + 5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
precursor tRNA + H2O
mature tRNA + 5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
RNA
5'-phosphomononucleotides
show the reaction diagram
-
3' to 5'direction only
-
-
?
RNA
nucleoside 5'-monophosphate
show the reaction diagram
-
-
-
-
?
RNA + H2O
phosphomononucleotides
show the reaction diagram
-, Q88DE8
RNA turnover
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
ss RNA + H2O
5'-phosphomononucleotides
show the reaction diagram
-
-
-
?
tRNAiMet + H2O
?
show the reaction diagram
P39112, -
complete degradation of the hypomodified tRNA requires both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 fails to degrade tRNAi Met, showing this to be a specific Rrp44 substrate
-
-
?
additional information
?
-
-
Dis3 is responsible for exosome core activity
-
-
-
additional information
?
-
P37202
RNase activity of Dis3 is required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Dis3 is suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region
-
-
-
additional information
?
-
-
the enzyme is involed in processing of polycistronic tRNA transcripts. Polynucleotide phosphorylase (PNPase) and RNase II are required for the removal of the 3 Rho-dependent terminator sequences
-
-
-
additional information
?
-
-, Q88DE8
mRNA is a direct substrate for RNase R
-
-
-
additional information
?
-
-, Q88DE8
RNase R is a 3'-5'-exoribonuclease that is very processive and can efficiently digest RNAs having extensive secondary structures, such as rRNA, RNAs containing repetitive extragenic palindromic (REP) sequences, or the transfer-messenger RNA required for trans-translation.
-
-
-
additional information
?
-
-, Q88DE8
RNase R is also involved in the processing of 16S and 5S rRNA
-
-
-
additional information
?
-
-, Q88DE8
The absence of RNase R leads to moderate increases in the mRNA levels of some RNases and RNA helicases, but other RNases and RNA helicases are not affected.
-
-
-
additional information
?
-
-
at optimal (37C) or elevated (42C) growth temperatures, the loss of RNase R in the RNase R mutant has no major consequence on bacterial growth and has a moderate impact on normal gene regulation. At lower temperatures (25C or 30C), the loss of RNase R has a significant impact on bacterial growth and results in the accumulation of structured RNA degradation products. Concurrently, gene regulation is affected and specifically results in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R is sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant is independent of previously identified competence regulators. The rnr mutant is competent for genetic transformation. RNase R is dispensable for the intracellular multiplication of Legionella pneumophila in both human and protozoan hosts. A physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability
-
-
-
additional information
?
-
-
RNase II is one of the major enzymes involved in mRNA processing. If the CSD is limiting the action of RNase II in vivo, it may play an important role working as a brake and thus preventing the massive degradation of RNA
-
-
-
additional information
?
-
-
RNase II degrades RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. RNase II activity is impaired by double-stranded RNAs
-
-
-
additional information
?
-
Legionella pneumophila JR32
-
at optimal (37C) or elevated (42C) growth temperatures, the loss of RNase R in the RNase R mutant has no major consequence on bacterial growth and has a moderate impact on normal gene regulation. At lower temperatures (25C or 30C), the loss of RNase R has a significant impact on bacterial growth and results in the accumulation of structured RNA degradation products. Concurrently, gene regulation is affected and specifically results in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R is sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the rnr mutant is independent of previously identified competence regulators. The rnr mutant is competent for genetic transformation. RNase R is dispensable for the intracellular multiplication of Legionella pneumophila in both human and protozoan hosts. A physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability
-
-
-
additional information
?
-
Escherichia coli SK4803
-
RNase II is one of the major enzymes involved in mRNA processing. If the CSD is limiting the action of RNase II in vivo, it may play an important role working as a brake and thus preventing the massive degradation of RNA
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
K+
-
necessary for activity
K+
-
activating
K+
-
activation against poly(A), -(U), -(C), RNA and T4 mRNA
K+
-
activating; maximum concentration for activation: 0.15 mM at pH 7.4 and 8.0
K+
-
necessary for activity; optimum for poly(A) as substrate: 100 mM; optimum for poly(C) as substrate: 25 mM
K+
-
100 mM KCl stimulating
K+
-
required
Li+
-
can substitute for K+ to a small extend
Mg2+
-
necessary for activity
Mg2+
-
necessary for activity; optimum: 2 mM
Mg2+
-
necessary for activity; optimum: 1 mM
Mg2+
-
necessary for activity; optimum: 1.5 mM
Mg2+
-
necessary for activity; optimum: 1-2 mM
Mg2+
-
necessary for activity
Mg2+
-
necessary for activity
Mg2+
-
increases activity
Mg2+
-
required
Mg2+
-
required, 5-20 mM optimal concentration
Mg2+
-
for catalysis of mRNA but not for substrate binding
Mg2+
-
Asp209, but not Asp207, contacting Mg2+ in addition to Asp201, Asp210, and the phosphate group of nt 13
Mg2+
-
is important for substrate binding of RNase R in the channel as well as for catalysis. Asp278 is predicted to coordinate a Mg2+ at the catalytic center of RNase R
Mg2+
-
required for the catalysis
Mg2+
-
dependent on
Mn2+
-
necessary for activity
Mn2+
-
can partially replace Mg2+
Mn2+
-
necessary for activity; optimum: 0.3 mM, stimulation about two-thirds the rate with Mg2+
Mn2+
-
can partially replace Mg2+
Mn2+
-
increases activity
Na+
-
increases activity against T4 mRNA, can substitute for K+ in activation against T4 mRNA and RNA
NH4+
-
activating; can substitute for K+ to a small extend
NH4+
-
activating; stimulates at pH 9.5 about 100%, optimum: 50 mM
NH4+
-
can substitute for K+ to a small extend
Zn2+
-
induces a folding reaction resulting in protein preparations with 50-60% alpha-helical content and 10-20% beta-sheet structure, critical concentration for refolding bigger or equal to 0.1 mM
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Ca2+
-
competitive inhibitor
DNA oligonucleotide with strong duplex structure and 3' single strand
-
-
-
DNA oligonucleotide with strong duplex structure and 5' single strand
-
-
-
DNA oligonucleotide with strong duplex structure, 3' and 5' single strand
-
-
-
DNA oligonucleotide with weak duplex structure and 3' single strand
-
-
-
DNA oligonucleotide with weak duplex structure and 5' single strand
-
-
-
DNA oligonucleotide with weak duplex structure, 3' and 5' single strand
-
-
-
DNA stem-loop structure
-
free 3'- and 5'-arms needed for potent inhibition, weaker stem-loops are better inhibitors than their counterpart with a strong duplex
-
ds plasmid DNA
-
-
-
EDTA
-
metal binding
EDTA
-
complete inhibition
mixed RNA-DNA oligonucleotides
-
-
-
monovalent or divalent cations
-
high concentrations
-
monovalent or divalent cations
-
10 mM of Mg2+ reduce activity 2fold, activity barely detectable at 20 mM Mg2+; high concentrations
-
Na+
-
substrate: artificial polynucleotides
NaCl
-
a salt concentration of 0.05 M inhibits 50% of the activity, salt optimum: 0.01 M
oligonucleotides
-
competitive inhibition, fragments bind without being destroyed; small to medium sized
p-chloromercuribenzoate
-
-
poly(A) binding protein
-
inhibits degradation of poly(A)+-strands
-
poly(dC)
-
19 to 29 monomer units chain lenght
Poly(U)
-
strong competitive inhibitor of T4 mRNA-degradation
RNA stem-loop structure
-
built by repetitive extragenic palindromic sequences; the lower the stability of the RNA-stem, the faster the degradation
-
RNA with a 2',3'-cyclic phosphate group
-
on the potential substrate molecule
-
RNA with a terminal 3'-phosphate group
-
on the potential substrate molecule
RNA with a terminal 3'-phosphate group
-
on the potential substrate molecule
RNA with a terminal 3'-phosphate group
-
on the potential substrate molecule
Rna with a terminal 5'-phosphate group
-
-
rRNA
-
weak inhibitor of T4 mRNA-degradation
SDS
-
complete inhibition 10 s after addition of SDS
spermine
-
slight inhibition of poly(U)-degradation
ss DNA oligonucleotides
-
competitive inhibition, potent inhibitors have a poly(dC)-chain length of 23-29
-
ss DNA oligonucleotides
-
-
-
ss DNA oligonucleotides
-
-
-
Sucrose
-
slight inhibition, in 1% and 5% sucrose 5% and 20% of the activity inhibited
thymidine nucleotides
-
potent inhibitor, pTp at a concentration of 0.00004 M inhibits degradation of poly(A) by 60%, pTpT at a concentration of 0.0005 M inhibits degradation of poly(A) by 30%
-
Urea
-
in 0.5 and 0.05 M urea 58% and 11% of the activity inhibited, complete inhibition at 1 M urea
Urea
-
loss of 40% of activity in presence of 1.6 M, 60% in presence of 2.4 M and 75% in presence of 3.2 M urea
Zn2+
-
competitive inhibitor
monovalent or divalent cations
-
high concentrations; Mg2+-concentrations above 2 mM
-
additional information
-
not inhibited by RNase A-type inhibitor
-
additional information
-
RNase II activity is blocked by the presence of double-stranded structures on the RNA molecule
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
cadaverine
-
can replace missing Mg2+ to a small extent
IPTG
-
1 mM, after 2 h RNase major protein in cell extracts of E.coli strain BL21(DE3)
putrescine
-
can replace missing Mg2+ to a small extent
-
spermidine
-
can replace missing Mg2+ to a small extent
spermine
-
can replace missing Mg2+ to a small extent
spermine
-
activates in absence of K+ the poly(C)- and poly(A)-breakdown
spermine
-
stimulating
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0003
-
Poly(A)
-
35-nt poly(A), mutant E542A, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
0.00125
-
Poly(A)
-
35-nt poly(A), wild-type, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
7.5e-05
-
Poly(U)
-
-
0.0004
-
polyadenosine
-
pH 7.2, 23C
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.41
-
Poly(A)
-
35-nt poly(A), wild-type, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
80800
-
Poly(A)
-
35-nt poly(A), mutant E542A, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
230
-
Poly(A)
-
35-nt poly(A), wild-type, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
15661
236
-
Poly(A)
-
35-nt poly(A), mutant E542A, at 37C, in 20 mM Tris-HCl buffer, pH 8, 100 mM KCl, 1 mM MgCl2, and 1 mM dithiothreitol
15661
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0037
-
DNA oligonucleotide with strong duplex structure and 3' single strand
-
pH 7.2, 23C
-
0.0331
-
DNA oligonucleotide with strong duplex structure and 5' single strand
-
pH 7.2, 23C
-
0.0014
-
DNA oligonucleotide with strong duplex structure, 3' and 5' single strand
-
pH 7.2, 23C
-
0.0008
-
DNA oligonucleotide with weak duplex structure and 3' single strand
-
pH 7.2, 23C
-
0.0336
-
DNA oligonucleotide with weak duplex structure and 5' single strand
-
pH 7.2, 23C
-
0.0005
-
DNA oligonucleotide with weak duplex structure, 3' and 5' single strand
-
pH 7.2, 23C
-
10
-
EDTA
-
malE-malF mRNA transcripts incubated at 37C
0.05
-
NaCl
-
salt 0.01 M
0.0005
-
polydeoxycytidine
-
29 monomer units, pH 7.2, 23C
-
0.0007
-
polydeoxycytidine
-
25 monomer units, pH 7.2, 23C
-
0.0015
-
polydeoxycytidine
-
23 monomer units, pH 7.2, 23C
-
0.0057
-
polydeoxycytidine
-
21 monomer units, pH 7.2, 23C
-
0.0148
-
polydeoxycytidine
-
19 monomer units, pH 7.2, 23C
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.004
-
-
substrate: poly(A)
0.04
-
-
activity of the most active fraction, substrate: T4 mRNA
970
-
-
pH 8.0, 37C
additional information
-
-
95600 units/mg: one unit is defined as the amount of enzyme producing 0.1 A260 acid-soluble materials in 3 h at 37C
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.4
-
-
minor pH optimum
7.5
9
-
-
8
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
9
-
11% of the optimal activity observed at pH 6.0, 55% at pH 9.0
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
assay at
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
-
Cajal bodies
-
Manually annotated by BRENDA team
additional information
-
protein TbMP42
-
Manually annotated by BRENDA team
additional information
Q08162
exosome, i.e. a macromolecule complex involved in RNA degradation
-
Manually annotated by BRENDA team
additional information
Trypanosoma brucei 427
-
protein TbMP42
-
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
33000
-
-
SDS-PAGE
35000
-
-
gel filtration
37000
-
-
native state gel filtration
65000
-
-
sucrose density gradient centrifugation
70000
-
-
SDS-PAGE
70000
-
-
-
80000
90000
-
native and SDS-PAGE, gel filtration
80000
-
-
SDS-PAGE
83000
-
-
gel filtration
88000
-
-
gel filtration
92000
-
-
SDS-PAGE
160000
-
-
SDS-PAGE
175000
-
-
amino acid sequence
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 12967, MALDI-TOF mass spectrometry; x * 13000, SDS-PAGE
monomer
-
1 * 70000
additional information
-
human Rrp44 proteins shows an overall structure similar to that of Escherichia coli RNase II
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
native RNase II and its RNA-bound complex
-
three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single stranded RNA
-
Rrp44 in complex with single-stranded RNA, to 2.3 A resolution. Structure of Rrp44 displays CSD1, CSD2, RNB, and S1 domains. The two N-terminal cold shock domains (CSD1, residues 271-399; CSD2, residues 400-475) and the C-terminal S1 domain (residues 911-998) display characteristic OB folds, with five antiparallel beta strands organized in a beta barrel structure. CSD1 is fused to an N-terminal alpha helix (residues 261-268) and the S1 domain has an insertion of three beta strands (between beta3 and beta4) as compared to a standard OB fold. The RNB domain is centered around a core and is surrounded by several alpha helices
Q08162
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
for 5 min, in absence of substrate, less than 1% of activity remains; in presence of substrate, product or deoxy-oligonucleotides (dC)27: active up to 60 min
50
-
-
in 0.1 M Tris-Cl, pH 8.0, for 15 min, loss of 20-30% of activity; in 0.1 M Tris-Cl, pH 8.0, for 60 min, loss of 40-60% of activity
60
-
-
for 1 min completely destroys enzyme activity
90
-
-
for 1 min, no activity remains
additional information
-
-, Q88DE8
Following cell growth at 10C by counting viable cells, rather than by turbidity, yields very similar growth rates. The doubling times are about 7 h for the wildtype strain and 15.5 h for the rnr mutant strain.; In LB medium, inactivation of the rnr gene impairs growth at 4C, but not at 30C (dispensability of RNase R). Other nucleases can perform its role under these conditions; Significant reduction of the growth rate at 10C: the doubling time increased from 6 h in the wild-type strain to 16.5 h in the rnr mutant strain.; The expression of the rnr gene increases less than twofold after a 2-h cold shock in which the culture temperature is decreased from 30C to 10C.
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, lyophilized purified enzyme, addition of 25 mg of bovine serum mercaptalbumin, stable for at least 2 months
-
-20C, small aliquots of purified enzyme, 10% glycerol, stable for at least 19 months
-
-70C, stable for at least 1 month without loss of activity
-
0C, half-life of 5-7 days
-
deep frozen, lyophilized crude enzyme, several months, stable
-
frozen, crude enzyme, 1 month, stable
-
4C, 10-ml fractions containing 1 mg trypsin inhibitor carrier protein because RNase II loses activity under this conditions at protein concentrations below 0.1 mg/ml
-
-10C, 1.5 ml aliquots, 1 mg of protein per ml, 6 months, small decrease of activity
-
-70C, more than 50% of activity after more than a year
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purified on protein A-agarose and affinity GST-AtRrp4p inmobilized on nitrocellulose
-
partial purification that includes DEAE-Sepharose chromatography
-
purification includes M2-agarose affinity resin
-
all mutants, with the exception of R500K, by histidine affinity chromatography and the AKTA fast protein liquid chromatography system
-
by affinity chromatography
-
by centrifugation, ion exchange and hydrophobic interaction chromatography
-
Escherichia coli strain BL21(DE3)
-
partial purification, between 50-100fold
-
RNase R and RNase II constructs, full-length wild-type RNase R and RNase R mutant D278N
-
to apparent homogeneity, 270fold
-
wild-type and RNase II mutants purified by affinity chromatography
-
5300-26500fold, to homogeneity
-
partial purification, 500-700fold
-
purified by inmunoprecipitation
-
370fold, nearly to homogeneity
-
full-length protein and mutants purified by affinity Ni-NTA chromatography, followed by ion exchange chromatography and gel filtration. 242-1001 construct purified to homogeneity
Q08162
includes glutathione-Sepharose resin chromatography
-
Rrp44-exosome (RE) architecture suggests an active site sequestration mechanism for strict control of 3' exoribonuclease activity in the RE complex
-
by histidine affinity chromatography and an AKTA HPLC system
-
by histidine affinity chromatography and an AKTA HPLC system
C6GKN7
anion exchange chromatography at denaturing conditions, in some cases followed by isoelectric focusing and dye binding chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli as a glutathione-S-transferasa fusion protein
-
expressed in Drosophila cell line
-
Escherichia coli strain JM109
-
pACYC184 derivative expressing RNase II, RNase II lacking cold shock domain-1 or mutant D209N under araBAD promoter. PACYC184 derivative expressing RNase R under araBAD promoter. RNase II and mutant D209N overexpressed from pET plasmid constructs in CH12 DELTArna cells
-
RNase II wild-type, mutant and truncated proteins, cloned into plasmid pFCT6.1, overexpressed in Escherichia coli BL21(DE3)
-
RNase R and RNase II constructs cloned into vector pET44R and overexpressed in Escherichia coli BL21II-R-(DE3)pLysS
-
vapour-diffusion method, wild-type RNase II is crystallized in two crystal forms, both of which belonged to space group P2(1). X-ray diffraction data are collected to 2.44 and 2.75 A resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 A, beta = 111.9 and a = 119.6, b = 57.2, c = 121.2 A, beta = 99.7, respectively. The RNase II D209N mutant gives crystals that belonged to space group P6(5), with unit-cell parameters a = b = 86.3, c = 279.2 A, and diffract to 2.74 A
-
wild-type and mutants overexpressed from pFCT6.9 vector as His6-tagged fusion proteins in Escherichia coli BL21(DE3)
-
wild-type and RNase II mutants cloned into plasmid pFCT6.9 and expressed in Escherichia coli BL21(DE3)
-
X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of RNase II. Structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity
-
DNA fragments corresponding to the entire open reading frame in each gene are subcloned into a TA cloning vector
-
expressed in Escherichia coli strain BL21(DE3)
-
Expression of rnr mutant (inactivation of Rnase R) is performed in Escherichia coli. Overexpression of genes corresponding to the flagellar apparatus or to the biosynthesis of cofactors by the absence of RNase R gene.
-, Q88DE8
expressed in Escherichia coli (XL-1)
-
expression in Escherichia coli
-
full-length Rrp44 (residues 1-1001) and truncated constructs of Rrp44 expressed from a pETM11 vector in Escherichia coli
Q08162
wild-type Rrp44, Rrp44-20, and Rrp44-cat are expressed in Escherichia coli as GST fusions
P39112, -
into the pET-15b vector, cloned into Escherichia coli DH5alpha, subsequently transformed into Escherichia coli strain BL21(DE3)
-
into the pET-15b vector, transformed into Escherichia coli Novablue, subsequently transformed into Escherichia coli strain BL21(DE3)
C6GKN7
expression in Escherichia coli M15pREP4
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D155M
-
truncated RNase II protein pETIIDELTACSD1DELTAS1 consisting of the nuclease domain alone, but lacking any part of CSD2. Removal of the RNA-binding domains does allow RNase II to proceed further
D201N
-
significant loss of activity in degradation of poly(A) (0.2% of that of the wild-type enzyme). Generates a 10-11-nt fragment as a major degradation product, although longer reaction times result in the usual 4-nt fragment as a secondary product
D201N
-
activity is highly impaired, 0.2% of the specific activity of wild-type enzyme
D201N
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D201N/E390A
-
very similar specific specific activity to the wild-type
D201N/E390A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D201N/Y313F
-
shows less than 0.1% of the specific activity present in the wild-type
D201N/Y313F
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D201N/Y313F/E390A
-
shows less than 0.1% of the specific activity present in the wild-type
D201N/Y313F/E390A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D207N
-
still retains 12% activity
D207N
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D209N
-
has less than 1% of the wild-type RNase activity, has similar affinities for the RNA substrate as the wild-type enzyme
D209N
-
catalytically inactive, is unable to complement RNase II deletion
D209N
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D210N
-
significant loss of activity in degradation of poly(A) (0.3% of that of the wild-type enzyme). Generates a 10-11-nt fragment as a major degradation product, although longer reaction times result in the usual 4-nt fragment as a secondary product
D210N
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
D278N
-
mutation at the catalytic center of RNase R, is inactive on A(4), but retains 4% activity of wild-type RNase R on poly(A) and A(17)
E390A
-
specific activity is very similar to that of the wild-type
E390A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
E542A
-
extraordinary catalysis and binding abilities that turns RNase II into a super-enzyme. More than a 100fold increase in the specific exoribonucleolytic activity, significantly increases affinity for the poly(A) substrate
E542A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
F358A
-
the protein is 2fold more active than the wild-type
R500A
-
shows more than a 40000fold reduction in specific activity when compared with the wild-type
R500A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
R500K
-
shows less than 0.1% of the specific activity present in the wild-type
Y253A
-
26% of the activity of the enzyme persists, significantly impairs RNA binding
Y253A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
Y253A/F358A
-
12% of the activity of the enzyme persists, whereas RNA binding affinity is not significantly affected
Y253A/F358A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
Y313A
-
100fold reduction of specific activity
Y313A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
Y313F
-
specific activity is very similar to that of the wild-type
Y313F
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
Y313F/E390A
-
specific activity is not affected
Y313F/E390A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
C425A
-
cannot be classified as polymorphic in the Japanese population. In the Korean, Mongolian, Ovambo, Turkish, and German DNA no genotype other than homozygotic 425C allele in RNASE2 at each single nucleotide polymorphism site is found
D275A
-
is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence
D283R
-
is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence
D275A
Legionella pneumophila JR32
-
is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence
-
D283R
Legionella pneumophila JR32
-
is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence
-
A815F
Q08162
degrades RNA duplexes with 7 or 14 nucleotides of ssRNA overhang significantly slower (about 4fold) than the wild-type enzyme
A815W
Q08162
degrades RNA duplexes with 7 or 14 nucleotides of ssRNA overhang significantly slower (about 3fold) than the wild-type enzyme
D551N
P39112, -
mutation in Rrp44-cat abolishes the exonucleolytic activity of Rrp44 without affecting its ability to bind RNA
F358A
-
site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme
additional information
-
hybrid proteins are constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability are examined. Both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. The S1 domains investigated are not equivalent
additional information
-
construction of a large set of RNase II truncated proteins and comparison of them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants are determined using different single- or double-stranded substrates. The results obtained reveal that S1 is the most important domain in the establishment of stable RNAprotein complexes, and its elimination results in a drastic reduction on RNA-binding ability. The N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. The biochemical results obtained with a mutant that lacks both putative RNA-binding domains, reveals the presence of an additional region involved in RNA binding. Such region, is identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain
additional information
-
RNase RDELTACSDs is missing the first 221 amino acids of RNase R, which include CSD1 and CSD2. RNase RDELTABasic lacks the 83 amino acids from the C terminus, which comprise the low complexity, highly basic region. RNase RDELTAS1 is truncated 170 amino acids from the C-terminus to remove both the S1 domain and the low complexity, highly basic region. RNase RDELTACSDsDELTAS1 consists of the nuclease domain alone, and, therefore, lacks all of the putative RNA-binding domains. Decrease in affinity upon deletion of either the CSDs or the S1 domain. RNase RDELTABasic displays 2fold higher activity than full-length wild-type RNase R. RNase RDELTACSDs looses 30% of the activity of full-length RNase R on poly(A) 90% on the shorter A(17) substrate. The RNase RDELTACSDsDELTAS1 truncated protein retains only 0.5% activity of the full-length protein on poly(A), and only 0.02% activity on A(17). All of the RNase R-truncated proteins have comparable activity on A(4)
additional information
-
DELTACSDb and DELTAS1b mutants, are more than 90% soluble. Similarly, the solubility of the RNB derivative, which lacks both putative RNA-binding domains, is also greater than 90%. The DELTACSDa mutant is only 60% soluble. Elimination of the whole CSD domain (DELTACSDb) or part of it (DELTACSDa) does not affect the exonucleolytic activity of RNase II, and even improves its activity significantly
D209N
Escherichia coli SK4803
-
has less than 1% of the wild-type RNase activity, has similar affinities for the RNA substrate as the wild-type enzyme
-
additional information
Escherichia coli SK4803
-
DELTACSDb and DELTAS1b mutants, are more than 90% soluble. Similarly, the solubility of the RNB derivative, which lacks both putative RNA-binding domains, is also greater than 90%. The DELTACSDa mutant is only 60% soluble. Elimination of the whole CSD domain (DELTACSDb) or part of it (DELTACSDa) does not affect the exonucleolytic activity of RNase II, and even improves its activity significantly
-
D551N
Q08162
abolishes the exonucleolytic activity
additional information
Q08162
Rrp44 242-1001 (Rrp44DELTAN) lacks the predicted N-terminal PIN domain, shows no detectable difference in activity toward ssRNA substrates as compared to recombinant Rrp44
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
degradation
-
RNA-binding domains of RNase II play a more important role in its exoribonuclease activity than they do in the activity of RNase R
medicine
-
new factor in the IFN-mediated antiviral barrier against HIV-1