BRENDA - Reference to 5.4.99.24; Id = 748788

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

D139N

site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive

Escherichia coli

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

23S rRNA uridine955/uridine2504/uridine2580

Escherichia coli

-

23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580

-

?

Escherichia coli

P0AA39

M15

-

23S rRNA uridine955/uridine2504/uridine2580

-

748788

Escherichia coli

23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580

-

?

ribosomal large subunit pseudouridine synthase C

UniProt

Escherichia coli

RLuc

-

Escherichia coli

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

D139N

site-directed mutagenesis of the chimeric enzyme mutant RluCD, the mutant is catalytically inactive

Escherichia coli

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

23S rRNA uridine955/uridine2504/uridine2580

Escherichia coli

-

23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580

-

?

23S rRNA uridine955/uridine2504/uridine2580

-

748788

Escherichia coli

23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580

-

?

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

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

748788

Leppik

Random pseuoduridylation in v ...

Escherichia coli

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45

6098-6108

2017

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1

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Toh

An indigenous posttranscriptio ...

Escherichia coli

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Identification of novel Escher ...

Escherichia coli

J. Bacteriol.

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Mizutani

Crystal structures of the cata ...

Escherichia coli

Biochemistry

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2004

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1

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1

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2

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5857

Corollo

-

Crystallisation and characteri ...

Escherichia coli

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55

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-

1

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1

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1

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1

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702195

Huang

Identification of two Escheric ...

Escherichia coli

Biochemistry

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15951-15957

1998

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1

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1

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704389

Conrad

The rluC gene of Escherichia c ...

Escherichia coli

J. Biol. Chem.

273

18562-18566

1998

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1

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1

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1

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1

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1

-

1

-

1

-

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

Data extracted from this reference: