Literature summary for 5.4.99.24 extracted from

Leppik, M.; Liiv, A.; Remme, J.
Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly (2017), Nucleic Acids Res., 45, 6098-6108 .

Search for more literature for 5.4.99.24

Cloned(Commentary)

Cloned (Comment)	Organism
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

Cloned (Comment)

Organism

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

Protein Variants

Protein Variants	Comment	Organism
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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
23S rRNA uridine955/uridine2504/uridine2580	Escherichia coli	-	23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0AA39	M15	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
23S rRNA uridine955/uridine2504/uridine2580	-	Escherichia coli	23S rRNA pseudouridine955/pseudouridine2504/pseudouridine2580	-	?

Synonyms

Synonyms	Comment	Organism
ribosomal large subunit pseudouridine synthase C	UniProt	Escherichia coli
RLuc	-	Escherichia coli

General Information

General Information	Comment	Organism
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