ABSTRACT The _Escherichia coli_ chromosome encodes toxin-antitoxin pairs. The toxin RelE cleaves mRNA positioned at the A-site in ribosomes, whereas the antitoxin RelB relieves the effect of

RelE. The hyperthermophilic archaeon _Pyrococcus horikoshii_ OT3 has the archaeal homologs aRelE and aRelB. Here we report the crystal structure of aRelE in complex with aRelB determined at

a resolution of 2.3 Å. aRelE folds into an α/β structure, whereas aRelB lacks a distinct hydrophobic core and extensively wraps around the molecular surface of aRelE. Neither component

shows structural homology to known ribonucleases or their inhibitors. Site-directed mutagenesis suggests that Arg85, in the C-terminal region, is strongly involved in the functional activity

more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS

OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A YOEB TOXIN CLEAVES BOTH RNA AND DNA Article

Open access 11 February 2021 MENT NUCLEOTIDYLTRANSFERASE TOXINS EXTEND TRNA ACCEPTOR STEMS AND CAN BE INHIBITED BY ASYMMETRICAL ANTITOXIN BINDING Article Open access 17 August 2023 SUBSTRATE

RECOGNITION AND CRYO-EM STRUCTURE OF THE RIBOSOME-BOUND TAC TOXIN OF _MYCOBACTERIUM TUBERCULOSIS_ Article Open access 12 May 2022 ACCESSION CODES ACCESSIONS PROTEIN DATA BANK * 1WMI

REFERENCES * Gerdes, K. Toxin-antitoxin modules may regulate synthesis of macromolecules during nutritional stress. _J. Bacteriol._ 182, 561–572 (2000). Article  CAS  Google Scholar  *

Galvani, C., Terry, J. & Ishiguro, E.E. Purification of the RelB and RelE proteins of _Escherichia coli_: RelE binds to RelB and to ribosomes. _J. Bacteriol._ 183, 2700–2703 (2001).

Article  CAS  Google Scholar  * Gotfredsen, M. & Gerdes, K. The _Escherichia coli relBE_ genes belong to a new toxin-antitoxin gene family. _Mol. Microbiol._ 29, 1065–1076 (1998).

Article  CAS  Google Scholar  * Christensen, S.K., Mikkelsen, M., Pedersen, K. & Gerdes, K. RelE, a global inhibitor of translation, is activated during nutritional stress. _Proc. Natl.

Acad. Sci. USA_ 98, 14328–14333 (2001). Article  CAS  Google Scholar  * Bech, F.W., Jorgensen, S.T., Diderichsen, B. & Karlstrom, O.H. Sequence of the relB transcription unit from

_Escherichia coli_ and identification of the _relB_ gene. _EMBO J._ 4, 1059–1066 (1985). Article  CAS  Google Scholar  * Masuda, Y., Miyakawa, K., Nishimura, Y. & Ohtsubo, E. _chpA_ and

_chpB_, _Escherichia coli_ chromosomal homologs of the _pem_ locus responsible for stable maintenance of plasmid R100. _J. Bacteriol._ 164, 1124–1135 (1993). Google Scholar  * Aizenman, E.,

Engelberg-Kulka, H. & Glaser, G. An _Escherichia coli_ chromosomal 'addiction module' regulated by guanosine 3′,5′-bispyrophosphate: a model for programmed bacterial cell

death. _Proc. Natl. Acad. Sci. USA_ 93, 6059–6063 (1996). Article  CAS  Google Scholar  * Grady, R. & Hayes, F. Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a

multidrug resistant, clinical isolate of _Enterococcus faecium_. _Mol. Microbiol._ 47, 1419–1432 (2003). Article  CAS  Google Scholar  * Pedersen, K., Christensen, S.K. & Gerdes, K.

Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. _Mol. Microbiol._ 45, 501–510 (2002). Article  CAS  Google Scholar  * Pedersen,

K. et al. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. _Cell_ 112, 131–140 (2003). Article  CAS  Google Scholar  * Christensen, S.K., Pedersen,

K., Hansen, G.H. & Gerdes, K. Toxin-antitoxin loci as stress-responsible-elements. ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. _J. Mol. Biol._ 332,

809–819 (2003). Article  CAS  Google Scholar  * Zhang, Y. et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in _Escherichia coli_. _Mol. Cell_ 12, 913–923

(2003). Article  CAS  Google Scholar  * Kamada, K., Hanaoka, F. & Burley, S.K. Crystal structure of the Maz/MazF complex: molecular bases of antidote-toxin recognition. _Mol. Cell_ 11,

875–884 (2003). Article  CAS  Google Scholar  * de la Cueva-Mendez, G. Distressing bacteria. Structure of a prokaryotic detox program. _Mol. Cell_ 11, 848–850 (2003). Article  CAS  Google

Scholar  * Christensen, S.K. & Gerdes, K. RelE toxins from bacteria and archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA. _Mol. Microbiol._ 48, 1389–1400 (2003).

Article  CAS  Google Scholar  * Takagi, H. et al. Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon _Pyrococcus horikoshii_ OT3. _Biochem. Biophys.

Res. Commun._ 319, 787–794 (2004). Article  CAS  Google Scholar  * Kakuta, Y. et al. Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from

hyperthermophilic archaeon _Pyrococcus horikoshii_ OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B. _Biochem. Biophys. Res. Commun._ 319, 725–732 (2004). Article 

CAS  Google Scholar  * Kawarabayashi, Y. et al. Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, _Sulfolobus tokodaii_ strain 7. _DNA Res._ 8, 123–140 (2001). Article 

Google Scholar  * Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. _J. Mol. Biol._ 233, 123–138 (1993). Article  CAS  Google Scholar  * Buckle, A.M.,

Schreiber, G. & Fersht, A.R. Protein-protein recognition: crystal structural analysis of a barnase–barstar complex at 2.0-Å resolution. _Biochemistry_ 33, 8878–8889 (1994). Article  CAS

  Google Scholar  * Kolade, O.O. et al. Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins. _Biochimie_ 84, 439–446 (2002). Article  CAS  Google

Scholar  * Graille, M., Mora, L., Buckingham, R.H., van Tilbeurgh, H. & Zamaroczy, M. Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein. _EMBO J._ 23,

1474–1482 (2004). Article  CAS  Google Scholar  * Landschulz, W.H., Johnson, P.F. & McKnight, S.L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. _Science_

243, 1681–1688 (1989). Article  CAS  Google Scholar  * Vinson, C.R., Sigler, P.B. & McKnight, S.L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins.

_Science_ 246, 911–246 (1989). Article  CAS  Google Scholar  * Weiss, M.A., Ellenberger, T., Wobbe, C.R., Lee, J. P, Harrison, S.C. & Struhl, K. Folding transition in the DNA-binding

domain of GCN4 on specific binding to DNA. _Nature_ 347, 575–578 (1990). Article  CAS  Google Scholar  * Ellenberger, T.E., Brandl, C.J., Struhl, K. & Harrison, S.C. The GCN4 basic

region leucine zipper binds DNA as a dimer of uninterrupted a helices: crystal structure of the protein–DNA complex. _Cell_ 71, 1223–1237 (1992). Article  CAS  Google Scholar  * Saudek, V.

et al. Solution structure of the basic region from the transcriptional activator GCN4. _Biochemistry_ 30, 1310–1317 (1991). Article  CAS  Google Scholar  * Nissen, P. et al. Crystal

structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. _Science_ 270, 1464–1472 (1995). Article  CAS  Google Scholar  * Song, H. et al. The crystal structure of human

eukaryotic release factor eRF1-mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. _Cell_ 100, 311–321 (2000). Article  CAS  Google Scholar  * Vestergaard, B. et al. Bacterial

polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1. _Mol. Cell_ 8, 1375–1382 (2001). Article  CAS  Google Scholar  * Selmer, M., Al-Karadaghi, S., Hirokawa, G.,

Kaji, A. & Liljas A. Crystal structure of Thermotoga maritime ribosome recycling factor: a tRNA mimic. _Science_ 286, 2349–2352 (1999). Article  CAS  Google Scholar  * Nakamura, Y. &

Ito, K. Making sense of mimic in translation termination. _Trends Biochem. Sci._ 28, 99–105 (2003). Article  CAS  Google Scholar  * Ito, K., Ebihara, K., Uno, M. & Nakamura, Y.

Conserved motifs of prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis. _Proc. Natl. Acad. Sci. USA_ 93, 5443–5448 (1996). Article  CAS  Google Scholar 

* Wilson, K.S. & Noller, H.F. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. _Cell_ 92, 131–139 (1998). Article  CAS 

Google Scholar  * Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. _Methods Enzymol._ 276, 307–326 (1997). Article  CAS  Google Scholar  *

Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. _Acta Crystallogr. D_ 55, 849–861 (1999). Article  CAS  Google Scholar  * Jones, T.A., Zou, J.Y., Cowan, S.W.

& Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. _Acta Crystallogr. A_ 47, 110–119 (1991). Article 

Google Scholar  * Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. _Acta Crystallogr. D_ 54, 905–921 (1998). Article 

CAS  Google Scholar  * Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. _J. Appl. Cryst._ 26,

283–291 (1993). Article  CAS  Google Scholar  * Wower, I.K., Zwieb, C.W., Guven, S.A. & Wower, J. Binding and cross-linking of tmRNA to ribosomal protein S1, on and off the _Escherichia

coli_ ribosome. _EMBO J._ 19, 6612–6621 (2000). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank N. Shimizu (Japan Synchrotron Radiation Research Institute) for

help with data collection using synchrotron radiation at beamline BL40B2. We are grateful to M. Ohara (Fukuoka) for helpful comments on the manuscript. This work was supported in part by a

grant from the National Project on Protein Structural and Functional Analyses from the Japan Ministry of Education, Culture, Sports, Science and Technology. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Bioscience and Biotechnology, Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, 812-8581, Japan Hisanori Takagi, 

Yoshimitsu Kakuta, Takahiro Okada & Makoto Kimura * Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810, Japan Min Yao & Isao Tanaka

Authors * Hisanori Takagi View author publications You can also search for this author inPubMed Google Scholar * Yoshimitsu Kakuta View author publications You can also search for this

author inPubMed Google Scholar * Takahiro Okada View author publications You can also search for this author inPubMed Google Scholar * Min Yao View author publications You can also search

for this author inPubMed Google Scholar * Isao Tanaka View author publications You can also search for this author inPubMed Google Scholar * Makoto Kimura View author publications You can

also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Makoto Kimura. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIG. 1 Viable count. (PDF 53 kb) SUPPLEMENTARY FIG. 2 Sequence comparison. (PDF 28 kb) SUPPLEMENTARY TABLE 1 Major interactions. (PDF 20

kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Takagi, H., Kakuta, Y., Okada, T. _et al._ Crystal structure of archaeal toxin-antitoxin RelE–RelB

complex with implications for toxin activity and antitoxin effects. _Nat Struct Mol Biol_ 12, 327–331 (2005). https://doi.org/10.1038/nsmb911 Download citation * Received: 02 December 2004 *

Accepted: 07 February 2005 * Published: 13 March 2005 * Issue Date: 01 April 2005 * DOI: https://doi.org/10.1038/nsmb911 SHARE THIS ARTICLE Anyone you share the following link with will be

able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing

initiative