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Escherichia coli K-12 substr. MG1655 Protein: RelB-RelE antitoxin/toxin complex and DNA-binding transcriptional repressor

Synonyms: RelBE

Subunit composition of RelB-RelE antitoxin/toxin complex and DNA-binding transcriptional repressor = [RelB]4[RelE]4
         RelB Qin prophage; antitoxin of the RelE-RelB toxin-antitoxin system and DNA binding transcriptional repressor = RelB
         Qin prophage; toxin of the RelE-RelB toxin-antitoxin system and cofactor to enhance the repressor activity of RelB = RelE (extended summary available)

Summary:
RelB is a DNA-binding transcriptional regulator which belongs to the ribbon-helix-helix (RHH) family of transcription factors [Anantharaman03, Cherny07, Li08] and it is an antitoxin that prevents the lethal action of the toxin [Gotfredsen98]. RelB is part of the relBE-hokD operon, which specifies a toxin-antitoxin system, and it is autoregulated by its own products, RelB and RelE [Gotfredsen98, Li08, Galvani01]. On the other hand, relE encodes a cytotoxin that is lethal or inhibitory to host cells [Gotfredsen98], and it also encodes a cofactor that enhances the repressor activity of RelB [Gotfredsen98, Li08, Galvani01].

The relBE-hokD operon is induced upon entering nutritional starvation conditions, such as the stringent response or acid starvation [Christensen01, Pistolese02], while the level of RelB antitoxin is reduced as a result of Lon-dependent proteolysis. Consequently, RelE toxin is liberated, leading to cell growth arrest and eventually cell death [Grady03]. The expression of the relBE-hokD operon under these conditions may have some as-yet-uncovered beneficial function [Gotfredsen98].

RelB and RelE form a high-affinity complex with a 2:1 stoichiometry when RelB is in excess [Overgaard08, Overgaard09a]. This interaction of RelE with RelB is essential for regulating the expression of the relBE-hokD operon and for neutralizing the toxic activity of RelE [Galvani01]. The ReB2-RelE complex represses transcription of the relBE-hokD operon [Li08] via strong cooperative binding to the relB promoter region [Gotfredsen98, Overgaard09a]. The 24-bp operator contains a hexad repeat (5'-[A/T]TGT[A/C]A-3') that is repeated twice on each strand [Li08, Li09, Overgaard09a]. The spacing between each half-site was found to be essential for cooperative interactions [Overgaard09a] between two RelB2-RelE heterotrimers. Only RelB makes contacts to the DNA and the RHH motif of RelB recognizes the four hexad repeats within the bipartite binding site. High affinity for DNA is only achieved in the presence of RelE [Li08], which stabilizes the tetrameric form of RelB [Li08].

When RelE is in excess, relBE transcription is stimulated [Overgaard08]. It has been suggested, that excess RelE leads to the formation of a RelE2-RelB2 complex that does not bind to the operator [Overgaard08, Overgaard09a].

RelB possesses a well-folded core domain at its N terminus followed by a flexible region at its C terminus, a pattern typical of other antitoxins [Li08]. The C terminus is responsible for dimerization of the dimeric core domain in the assembly of the RelB tetramer [Li08], and the N terminus is responsible for binding to the relB promoter region via its RHH domain [Overgaard08, Li08].

By using a low-toxicity mutant of RelE, RelER81A/R83A, the protein could be purified for structural studies. RelER81A/R83A exhibits an α/β-sandwich fold. Its C-terminal helix 4 lies next to a conserved positive charged cluster, the putative mRNA-binding site of RelE toxin. In a complex of RelER81A/R83A with a C-terminal peptide of RelB (RelBc), this helix is displaced by helix 3 of RelBc, resulting in the neutralization of the positively charged cluster of RelE [Li09].

The RelB-RelE complex in Pyrococcus horikoshii is heterotetrameric (RelB-RelE)2 [Takagi05], whereas in E. coli a RelB4-RelE4 complex may form a tight association with two adjacent binding sites on the promoter, which could involve either DNA bending or DNA-induced protein conformational change [Li08].

Several homologs have been identified on the chromosome of E. coli K-12 as well as on those of other organisms, such as Haemophilus influenzae and Vibrio cholerae, and in the E. coli plasmid P307 [Gotfredsen98]. Bacterial relBE systems are conserved in archaea, such as in Methanococcus jannaschii, Archaeoglobus fulgidus, and P. horikoshii OT3 [Gerdes00]. Alignment of the RelB homologs showed that these proteins are considerably more divergent than the RelE homologs [Gotfredsen98].

The sequence alignment of the RelB homologs shows that these proteins are considerably more divergent than the RelE homologs [Gotfredsen98].

Mutations in the relB gene confer a so-called delayed relaxed phenotype upon host cells [Lavalle65, Diderichsen77, Bech85], in which synthesis of stable RNA resumes approximately 10 min after the initiation of amino acid starvation [Gotfredsen98]. These findings provided the first sign that the stringent response system might be connected to the MazEF and RelBE systems through the translation apparatus [Wilson05].

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006276 - plasmid maintenance Inferred from experiment [Gotfredsen98]

DNA binding site length: 12 base-pairs

Symmetry: Inverted Repeat

Regulated Transcription Units (1 total): ?

Notes:

Credits:
Created 03-Apr-2009 by Santos-Zavaleta A , UNAM
Last-Curated ? 03-Apr-2009 by Santos-Zavaleta A , UNAM


Subunit of RelB-RelE antitoxin/toxin complex and DNA-binding transcriptional repressor: RelB Qin prophage; antitoxin of the RelE-RelB toxin-antitoxin system and DNA binding transcriptional repressor

Synonyms: RelB

Gene: relB Accession Numbers: EG10836 (EcoCyc), b1564, ECK1558

Locations: cytosol

Sequence Length: 79 AAs

Molecular Weight: 9.071 kD (from nucleotide sequence)

Molecular Weight: 9.0 kD (experimental) [Bech85]

GO Terms:

Biological Process: GO:0006351 - transcription, DNA-templated Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Gotfredsen98]
GO:0006355 - regulation of transcription, DNA-templated Inferred by computational analysis [UniProtGOA11a]
GO:0006950 - response to stress Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Galvani01]
GO:0003677 - DNA binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: cell processes protection cell killing
information transfer RNA related RNA degradation
information transfer RNA related Transcription related
regulation genetic unit regulated operon
regulation type of regulation posttranscriptional translation attenuation and efficiency
regulation type of regulation transcriptional level repressor

Unification Links: DIP:DIP-48258N , EcoliWiki:b1564 , PR:PRO_000023711 , Pride:P0C079 , Protein Model Portal:P0C079 , RefSeq:NP_416082 , SMR:P0C079 , String:511145.b1564 , UniProt:P0C079

Relationship Links: InterPro:IN-FAMILY:IPR007337 , PDB:Structure:2K29 , PDB:Structure:2KC8 , PDB:Structure:4FXE , Pfam:IN-FAMILY:PF04221

Essentiality data for relB knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]

Component enzyme of RelB-RelE antitoxin/toxin complex and DNA-binding transcriptional repressor : Qin prophage; toxin of the RelE-RelB toxin-antitoxin system and cofactor to enhance the repressor activity of RelB

Synonyms: RelE

Gene: relE Accession Numbers: EG11131 (EcoCyc), b1563, ECK1557

Locations: cytosol

Sequence Length: 95 AAs

Molecular Weight: 11.225 kD (from nucleotide sequence)

GO Terms:

Biological Process: GO:0006402 - mRNA catabolic process Inferred from experiment [Pedersen03]
GO:0017148 - negative regulation of translation Inferred from experiment [Christensen01, Pedersen02]
GO:0034198 - cellular response to amino acid starvation Inferred from experiment [Christensen01]
GO:0046677 - response to antibiotic Inferred from experiment [KolodkinGal09]
GO:0090502 - RNA phosphodiester bond hydrolysis, endonucleolytic Inferred from experiment [Hurley11, Pedersen03]
GO:0006351 - transcription, DNA-templated Inferred by computational analysis [UniProtGOA11a]
GO:0006355 - regulation of transcription, DNA-templated Inferred by computational analysis [UniProtGOA11a]
GO:0006950 - response to stress Inferred by computational analysis [UniProtGOA11a]
GO:0090305 - nucleic acid phosphodiester bond hydrolysis Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0004521 - endoribonuclease activity Inferred from experiment [Hurley11, Pedersen03]
GO:0005515 - protein binding Inferred from experiment [Galvani01]
GO:0043022 - ribosome binding Inferred from experiment [Galvani01]
GO:0070181 - small ribosomal subunit rRNA binding Inferred from experiment [Neubauer09]
GO:0003723 - RNA binding Inferred by computational analysis [UniProtGOA11a]
GO:0004518 - nuclease activity Inferred by computational analysis [UniProtGOA11a]
GO:0004519 - endonuclease activity Inferred by computational analysis [UniProtGOA11a]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
GO:0019843 - rRNA binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: cell processes protection cell killing
information transfer protein related translation
information transfer RNA related RNA degradation
regulation type of regulation posttranscriptional translation attenuation and efficiency

Unification Links: DIP:DIP-35978N , EcoliWiki:b1563 , Mint:MINT-1282331 , PR:PRO_000023712 , Pride:P0C077 , Protein Model Portal:P0C077 , RefSeq:NP_416081 , SMR:P0C077 , String:511145.b1563 , UniProt:P0C077

Relationship Links: InterPro:IN-FAMILY:IPR007712 , PDB:Structure:2KC8 , PDB:Structure:2KC9 , PDB:Structure:3KIQ , PDB:Structure:3KIS , PDB:Structure:3KIU , PDB:Structure:3KIX , PDB:Structure:4FXE , PDB:Structure:4FXH , PDB:Structure:4FXI , Pfam:IN-FAMILY:PF05016

Catalyzes:
an mRNA + H2O → a single-stranded RNA + a single-stranded RNA

Summary:
RelE is the toxin of the RelE-RelB toxin-antitoxin system [Gotfredsen98]. RelE inhibits protein translation by catalyzing cleavage of mRNA in the A site of the ribosome in response to amino acid starvation [Pedersen03, Christensen03, Neubauer09, Hurley11].

RelE is involved in regulation of cellular protein translation under nutritional stress conditions [Christensen01, Pedersen03, Christensen03]. RelE-mediated translation inhibition is reported to cause reversible inhibition of cell growth [Pedersen02]. The activity of tmRNA counteracts RelE translation inhibition [Pedersen03, Christensen03]. RelE and RF1 functionally interact [DiagoNavarro09].

RelE and RelB physically interact [Galvani01]. When cells are starved of amino acids, Lon protease degrades RelB; RelB degradation frees RelE and derepresses transcription of relBE. RelE accumulates in excess compared with its RelB antitoxin, and this free RelE causes translation inhibition [Christensen01]. Conversely, RelB binding to RelE protects RelB from proteolytic degradation [Cherny07, Overgaard09a]. The structure of the RelE-RelB complex and cooperative binding to the relBE promoter region has been studied; at low levels, RelE enhances the interaction between RelB and the promoter, while excess RelE destabilizes promoter binding of the complex [Li08, Overgaard08].

Solution structures of a RelE mutant together with a RelB peptide have been determined; RelB appears to induce conformational changes in the RelE active site, inactivating it [Li09]. Crystal structures of RelE alone and bound to a heterologous ribosome have elucidated the reaction mechanism of RelE. RelE binds to the ribosomal A site and cleaves mRNA after the second nucleotide of the codon; cleavage is ribosome-dependent [Neubauer09]. The in vivo frequency and codon specificity of RelE cleavage has been mapped; contrary to earlier in vitro results, no sequence preference can be seen in vivo. RelE appears to cleave within the first 100 codons in the coding region of mRNAs [Hurley11].

Mutations in the relB locus were initially identified by a "delayed relaxed" phenotype, characterized by a ~10 minute lag period followed by continued stable RNA synthesis in response to amino acid starvation [Lavalle65, Diderichsen77]. This phenotype is due to destabilization of RelB, resulting in hyperactivation of RelE [Christensen04].

relBE is overexpressed in persister cells. Ectopic expression of RelE increases the number of persister cells that survive normally lethal antibiotic treatment [Keren04]. Conversely, deletion of relBE was reported to increase survival after antibiotic treatment [KolodkinGal09].

Phylogenetic analysis of toxin-antitoxin systems has been performed [Anantharaman03, Pandey05].

Reviews: [Yamaguchi11, Yamaguchi09, Lewis08, Condon06, Lewis05a, Hayes03, Gerdes00]

Citations: [ChristensenDals08]

Essentiality data for relE knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]

References

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Bech85: Bech FW, Jorgensen ST, Diderichsen B, Karlstrom OH (1985). "Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene." EMBO J 4(4);1059-66. PMID: 2990907

Cherny07: Cherny I, Overgaard M, Borch J, Bram Y, Gerdes K, Gazit E (2007). "Structural and thermodynamic characterization of the Escherichia coli RelBE toxin-antitoxin system: indication for a functional role of differential stability." Biochemistry 46(43);12152-63. PMID: 17924660

Christensen01: Christensen SK, Mikkelsen M, Pedersen K, Gerdes K (2001). "RelE, a global inhibitor of translation, is activated during nutritional stress." Proc Natl Acad Sci U S A 98(25);14328-33. PMID: 11717402

Christensen03: Christensen SK, Gerdes K (2003). "RelE toxins from bacteria and Archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA." Mol Microbiol 48(5);1389-400. PMID: 12787364

Christensen04: Christensen SK, Gerdes K (2004). "Delayed-relaxed response explained by hyperactivation of RelE." Mol Microbiol 53(2);587-97. PMID: 15228536

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DiagoNavarro09: Diago-Navarro E, Mora L, Buckingham RH, Diaz-Orejas R, Lemonnier M (2009). "Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE." Mol Microbiol 71(1);66-78. PMID: 19019162

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

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Gotfredsen98: Gotfredsen M, Gerdes K (1998). "The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family." Mol Microbiol 29(4);1065-76. PMID: 9767574

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Li09: Li GY, Zhang Y, Inouye M, Ikura M (2009). "Inhibitory mechanism of Escherichia coli RelE-RelB toxin-antitoxin module involves a helix displacement near an mRNA interferase active site." J Biol Chem 284(21);14628-36. PMID: 19297318

Neubauer09: Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, Brodersen DE (2009). "The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE." Cell 139(6);1084-95. PMID: 20005802

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Pedersen03: Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, Ehrenberg M (2003). "The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site." Cell 112(1);131-40. PMID: 12526800

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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