Structural studies of the toxin-antitoxin proteins RelE and RelB from E. coli

TY - CONF

T1 - Structural studies of the toxin-antitoxin proteins RelE and RelB from E. coli

AU - Andersen, Kasper Røjkjær

AU - Overgaard, Martin

AU - Gerdes, Kenn

AU - Brodersen, Ditlev Egeskov

PY - 2006

Y1 - 2006

N2 - The bacterial toxin-antitoxin systemThe relBE operon in E. coli encodes two small proteins: A toxin, RelE (12 kDa) and an antitoxin, RelB (9 kDa). RelE is activated under nutritional stress and is able to inhibit protein synthesis by cleaving the mRNA in the ribosomal A-site. This stress response serves to down-regulate metabolism in the cell when growth conditions are limited. RelB is expressed in excess over RelE during balanced growth, and inhibits the toxicity of RelE by forming an extremely stable toxin-antitoxin complex. The activation of RelE is induced when the labile RelB protein is degraded by the Lon protease which is upregulated under stress conditions. RelE is then released from the tight complex and promotes mRNA degradation on the ribosome. The ribosomes will stall at positions where the mRNAs are damaged and can only be released through the transtranslation system, involving the special tRNA-mRNA mimic, tmRNA [1].Questions to be addressedMany questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from Pyrococcus horikoshii OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the relBE promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome.Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome.Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome. Crystals of RelB from E. coli We have recently obtained crystals of isolated RelB that crystallize in a tetragonal space group with cell parameters a=b=65.63 Å, c=38.51 Å. These crystals seem to contain both the full length protein and two well-defined degradation products. This might interestingly enough reflect the biology of RelB which is degraded before RelE can be released. But the true nature of these degradation products and their relation to the in vivo degradation of RelB has to be investigated further.

AB - The bacterial toxin-antitoxin systemThe relBE operon in E. coli encodes two small proteins: A toxin, RelE (12 kDa) and an antitoxin, RelB (9 kDa). RelE is activated under nutritional stress and is able to inhibit protein synthesis by cleaving the mRNA in the ribosomal A-site. This stress response serves to down-regulate metabolism in the cell when growth conditions are limited. RelB is expressed in excess over RelE during balanced growth, and inhibits the toxicity of RelE by forming an extremely stable toxin-antitoxin complex. The activation of RelE is induced when the labile RelB protein is degraded by the Lon protease which is upregulated under stress conditions. RelE is then released from the tight complex and promotes mRNA degradation on the ribosome. The ribosomes will stall at positions where the mRNAs are damaged and can only be released through the transtranslation system, involving the special tRNA-mRNA mimic, tmRNA [1].Questions to be addressedMany questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from Pyrococcus horikoshii OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the relBE promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome.Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome.Many questions remain to be answered in the bacterial toxin-antitoxin system. The crystal structure of RelBE from OT3 was previously solved at 2.3Å [2]. This structure shows the molecule in an inactive state, but how do these proteins look when they are separate? It is likely that RelE changes conformation upon release of RelB. We wish to answer these questions by solving the structure of both RelB and RelE alone. Biochemical evidences show that both RelB and RelBE complex auto-regulate their own promoter, but how is this performed? In order to address this from a structural point of view we are currently trying to form and crystallize a complex between either isolated RelB or RelBE and a part of the promoter region. Ultimately it will be of great interest to investigate how RelE recognises and cleaves the mRNA? In the attempt to investigate this, binding studies and crystallizations trials are performed with the small 30S ribosomal subunit and the entire 70S ribosome. Crystals of RelB from E. coli We have recently obtained crystals of isolated RelB that crystallize in a tetragonal space group with cell parameters a=b=65.63 Å, c=38.51 Å. These crystals seem to contain both the full length protein and two well-defined degradation products. This might interestingly enough reflect the biology of RelB which is degraded before RelE can be released. But the true nature of these degradation products and their relation to the in vivo degradation of RelB has to be investigated further.

M3 - Poster

T2 - the 38th crystallographic meeting at Erice

Y2 - 9 June 2006 through 19 June 2006

ER -