BRENDA - Enzyme Database

Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly

Leppik, M.; Liiv, A.; Remme, J.; Nucleic Acids Res. 45, 6098-6108 (2017)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
5.4.99.23
recombinant expression of chimeric enzyme mutants RluCD in Escherichia coli strains M15 of CD204, expression of chimeric pseudouridine synthase RluCD, under control of arabinose inducible araBAD promoter, interferes with ribosome formation in wild-type M15 strain and strain CD204 lacking RluC and RluD, while cells transformed with catalytically inactive RluCD (RluCD D139N) and empty vector (pQE60), used as a controls, represent a normal ribosomal profile in sucrose density gradient. Strain CD204 contains a mutant form of RF-2 (D131Y) that suppresses ribosome assembly defect caused by the deletion of the RluD. Strain rluD114 where yfiI (RluD) gene has been disrupted with Km cassette is used as a parent strain to construct strain CD204
Escherichia coli
5.4.99.24
recombinant expression of chimeric enzyme mutants RluCD in Escherichia coli strains M15 and CD204, expression of chimeric pseudouridine synthase RluCD, under control of arabinose inducible araBAD promoter, interferes with ribosome formation in wild-type M15 strain and strain CD204 lacking RluC and RluD, while cells transformed with catalytically inactive RluCD (RluCD D139N) and empty vector (pQE60), used as a controls, represent a normal ribosomal profile in sucrose density gradient. Strain CD204 contains a mutant form of RF-2 (D131Y) that suppresses ribosome assembly defect caused by the deletion of the RluD. Strain rluD114 where yfiI (RluD) gene has been disrupted with Km cassette is used as a parent strain to construct strain CD204
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
5.4.99.23
D139N
site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive
Escherichia coli
5.4.99.23
additional information
construction of a chimeric pseudouridine synthase (RluCD) containing the N-terminal S4 domain of enzyme RluC (EC 5.4.99.24) and the C-terminal catalytic domain of enzyme RluD (EC 5.4.99.23). The chimeric mutant is able to introduce excessive pseudouridines into rRNA at non-native positions. The chimeric enzyme RluCD is used as a tool to study an effect of over-modification of rRNA on the ribosome biogenesis. Excessive pseudouridylation of 23S rRNA reduces progression of ribosome assembly during early or middle stages. A modification interference approach identifies the sites in 23S rRNA whose modification prevents ribosome assembly. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. RluCD isomerizes many uridines of rRNA in a non-specific manner. Induction of the RluCD at the exponential growth phase leads to severe inhibition of translation while transcription is only slightly affected
Escherichia coli
5.4.99.24
D139N
site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive
Escherichia coli
5.4.99.24
additional information
construction of a chimeric pseudouridine synthase (RluCD) containing the N-terminal S4 domain of enzyme RluC (EC 5.4.99.24) and the C-terminal catalytic domain of enzyme RluD (EC 5.4.99.23). The chimeric mutant is able to introduce excessive pseudouridines into rRNA at non-native positions. The chimeric enzyme RluCD is used as a tool to study an effect of over-modification of rRNA on the ribosome biogenesis. Excessive pseudouridylation of 23S rRNA reduces progression of ribosome assembly during early or middle stages. A modification interference approach identifies the sites in 23S rRNA whose modification prevents ribosome assembly. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. RluCD isomerizes many uridines of rRNA in a non-specific manner. Induction of the RluCD at the exponential growth phase leads to severe inhibition of translation while transcription is only slightly affected
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
5.4.99.23
23S rRNA uridine1911/uridine1915/uridine1917
Escherichia coli
-
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
-
-
?
5.4.99.24
23S rRNA uridine955/uridine2504/uridine2580
Escherichia coli
-
23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
5.4.99.23
Escherichia coli
P33643
M15
-
5.4.99.24
Escherichia coli
P0AA39
M15
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5.4.99.23
23S rRNA uridine1911/uridine1915/uridine1917
-
748788
Escherichia coli
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
-
-
-
?
5.4.99.23
additional information
the recombinant chimeric enzyme mutant RluCD isomerizes many uridines of rRNA in a non-specific manner
748788
Escherichia coli
?
-
-
-
-
5.4.99.24
23S rRNA uridine955/uridine2504/uridine2580
-
748788
Escherichia coli
23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580
-
-
-
?
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
5.4.99.23
recombinant expression of chimeric enzyme mutants RluCD in Escherichia coli strains M15 of CD204, expression of chimeric pseudouridine synthase RluCD, under control of arabinose inducible araBAD promoter, interferes with ribosome formation in wild-type M15 strain and strain CD204 lacking RluC and RluD, while cells transformed with catalytically inactive RluCD (RluCD D139N) and empty vector (pQE60), used as a controls, represent a normal ribosomal profile in sucrose density gradient. Strain CD204 contains a mutant form of RF-2 (D131Y) that suppresses ribosome assembly defect caused by the deletion of the RluD. Strain rluD114 where yfiI (RluD) gene has been disrupted with Km cassette is used as a parent strain to construct strain CD204
Escherichia coli
5.4.99.24
recombinant expression of chimeric enzyme mutants RluCD in Escherichia coli strains M15 and CD204, expression of chimeric pseudouridine synthase RluCD, under control of arabinose inducible araBAD promoter, interferes with ribosome formation in wild-type M15 strain and strain CD204 lacking RluC and RluD, while cells transformed with catalytically inactive RluCD (RluCD D139N) and empty vector (pQE60), used as a controls, represent a normal ribosomal profile in sucrose density gradient. Strain CD204 contains a mutant form of RF-2 (D131Y) that suppresses ribosome assembly defect caused by the deletion of the RluD. Strain rluD114 where yfiI (RluD) gene has been disrupted with Km cassette is used as a parent strain to construct strain CD204
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
5.4.99.23
D139N
site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive
Escherichia coli
5.4.99.23
additional information
construction of a chimeric pseudouridine synthase (RluCD) containing the N-terminal S4 domain of enzyme RluC (EC 5.4.99.24) and the C-terminal catalytic domain of enzyme RluD (EC 5.4.99.23). The chimeric mutant is able to introduce excessive pseudouridines into rRNA at non-native positions. The chimeric enzyme RluCD is used as a tool to study an effect of over-modification of rRNA on the ribosome biogenesis. Excessive pseudouridylation of 23S rRNA reduces progression of ribosome assembly during early or middle stages. A modification interference approach identifies the sites in 23S rRNA whose modification prevents ribosome assembly. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. RluCD isomerizes many uridines of rRNA in a non-specific manner. Induction of the RluCD at the exponential growth phase leads to severe inhibition of translation while transcription is only slightly affected
Escherichia coli
5.4.99.24
D139N
site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive
Escherichia coli
5.4.99.24
additional information
construction of a chimeric pseudouridine synthase (RluCD) containing the N-terminal S4 domain of enzyme RluC (EC 5.4.99.24) and the C-terminal catalytic domain of enzyme RluD (EC 5.4.99.23). The chimeric mutant is able to introduce excessive pseudouridines into rRNA at non-native positions. The chimeric enzyme RluCD is used as a tool to study an effect of over-modification of rRNA on the ribosome biogenesis. Excessive pseudouridylation of 23S rRNA reduces progression of ribosome assembly during early or middle stages. A modification interference approach identifies the sites in 23S rRNA whose modification prevents ribosome assembly. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. RluCD isomerizes many uridines of rRNA in a non-specific manner. Induction of the RluCD at the exponential growth phase leads to severe inhibition of translation while transcription is only slightly affected
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
5.4.99.23
23S rRNA uridine1911/uridine1915/uridine1917
Escherichia coli
-
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
-
-
?
5.4.99.24
23S rRNA uridine955/uridine2504/uridine2580
Escherichia coli
-
23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580
-
-
?
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5.4.99.23
23S rRNA uridine1911/uridine1915/uridine1917
-
748788
Escherichia coli
23S rRNA pseudouridine1911/pseudouridine1915/pseudouridine1917
-
-
-
?
5.4.99.23
additional information
the recombinant chimeric enzyme mutant RluCD isomerizes many uridines of rRNA in a non-specific manner
748788
Escherichia coli
?
-
-
-
-
5.4.99.24
23S rRNA uridine955/uridine2504/uridine2580
-
748788
Escherichia coli
23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580
-
-
-
?
General Information
EC Number
General Information
Commentary
Organism
5.4.99.23
malfunction
excessive pseudouridylation of 23S rRNA by the chimeric mutant RluCD reduces progression of ribosome assembly during early or middle stages. Modification of sites in 23S rRNA prevents ribosome assembly, interfering positions are located inside the ribosome, mapping. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. Recombinant expression of RluCD protein itself inhibits ribosome assembly by binding to the precursor particles and blocking the assembly of r-proteins. The phenotypic effects are caused by the catalytic activity of the chimeric pseudouridine synthase RluCD, mechanism, overview. The excessive pseudouridines in rRNA species cause strong selection against 70S ribosome pool. Ribosome assembly defect causes degradation of unassembled rRNA and accumulation of small RNA fragments on the top of gradient
Escherichia coli
5.4.99.24
malfunction
excessive pseudouridylation of 23S rRNA by the chimeric mutant RluCD reduces progression of ribosome assembly during early or middle stages. Modification of sites in 23S rRNA prevents ribosome assembly, interfering positions are located inside the ribosome, mapping. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. Recombinant expression of RluCD protein itself inhibits ribosome assembly by binding to the precursor particles and blocking the assembly of r-proteins. The phenotypic effects are caused by the catalytic activity of the chimeric pseudouridine synthase RluCD, mechanism, overview. The excessive pseudouridines in rRNA species cause strong selection against 70S ribosome pool. Ribosome assembly defect causes degradation of unassembled rRNA and accumulation of small RNA fragments on the top of gradient
Escherichia coli
General Information (protein specific)
EC Number
General Information
Commentary
Organism
5.4.99.23
malfunction
excessive pseudouridylation of 23S rRNA by the chimeric mutant RluCD reduces progression of ribosome assembly during early or middle stages. Modification of sites in 23S rRNA prevents ribosome assembly, interfering positions are located inside the ribosome, mapping. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. Recombinant expression of RluCD protein itself inhibits ribosome assembly by binding to the precursor particles and blocking the assembly of r-proteins. The phenotypic effects are caused by the catalytic activity of the chimeric pseudouridine synthase RluCD, mechanism, overview. The excessive pseudouridines in rRNA species cause strong selection against 70S ribosome pool. Ribosome assembly defect causes degradation of unassembled rRNA and accumulation of small RNA fragments on the top of gradient
Escherichia coli
5.4.99.24
malfunction
excessive pseudouridylation of 23S rRNA by the chimeric mutant RluCD reduces progression of ribosome assembly during early or middle stages. Modification of sites in 23S rRNA prevents ribosome assembly, interfering positions are located inside the ribosome, mapping. It is plausible that pseudouridines can cause RNA misfolding when present at non-native positions. Recombinant expression of RluCD protein itself inhibits ribosome assembly by binding to the precursor particles and blocking the assembly of r-proteins. The phenotypic effects are caused by the catalytic activity of the chimeric pseudouridine synthase RluCD, mechanism, overview. The excessive pseudouridines in rRNA species cause strong selection against 70S ribosome pool. Ribosome assembly defect causes degradation of unassembled rRNA and accumulation of small RNA fragments on the top of gradient
Escherichia coli