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Tying up loose ends: Ribosome recycling in eukaryotes and archaea

Tying up loose ends: Ribosome recycling in eukaryotes and archaea. CNV meeting , july 2013. Elina Nürenberg. Tying up loose ends. termination. termination. initiation. initiation. elongation. elongation. r ibosome recycling. Physiological context: P rotein b iosynthesis .

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Tying up loose ends: Ribosome recycling in eukaryotes and archaea

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  1. Tying up loose ends:Ribosome recycling in eukaryotes and archaea CNVmeeting, july 2013 Elina Nürenberg

  2. Tying up loose ends termination termination initiation initiation elongation elongation ribosomerecycling

  3. Physiological context: Protein biosynthesis cytosole nucleus transcription Aa-tRNA, elongationfactors ABCE1 ? termination elongation recycling initiation mRNA, initiationfactors releasefactors eRF1 & eRF3 orABCE1 ? 60S 40S translation functionalprotein ribosomalsubunits polypeptide Release factor eRF1, ABCE1 Shoemaker et. al.2011 (yeast) Pestovaet. al. 2012 (human) Barthelme et. al. 2011 (archaea) folding

  4. Structure and function of ABCE1 P. abysii ABCE1 (PDB: 3BK7) • Highly conserved among eukaryotes and archaea, not present in bacteria • FeS-cluster domain (2 [Fe4S4]2+ ), two NBDs, 2 hinge regions • ATPase activity • Splitting of post-TC, vacant ribosomes and mRNA surveillance Karcheret. al. , 2008 Nürenberg& Tampé, 2013

  5. ABCE1 – Conformational dynamics • ATP binding/hydrolysis & FeS domain essential for ribosome splitting • Allosteric ATPase activation by eRF1 and the ribosome • MD simulation and modeling (in collaboration with Bert de Groot and Helmut Grubmüller, MPI of Biophysical Chemistry, Göttingen) Karcheret. al. , 2008 Nürenberg& Tampé, 2013

  6. Ribosome recycling: Mechanism (1) (1) • ABCE1 bindsatintersectionofribosomalsubunits • FeS domain binds C-domain of class 1 RFs • Unusual HLH motif and hinge regions are ribosomal binding sites Cryo-EM of stalled ribosomes Biochemical assays • NBDs functionallyasymmetric • ABCE1•AMPPNP bindstosmallribosomalsubunit (3) (2) Becker et. al. 2012 Nuerenberg & Tampé, 2013 Barthelme et. al. 2011

  7. At the interface of RNA and protein What is the conformational state of ABCE1 in… • Binding to post-termination complex? • Splitting the subunits? • Re-initiating a new translation cycle?

  8. Methods & Results Structure and conformational dynamics of ABCE1 in ribosome recycling Step 1: Arrest ABCE1 in different conformations by cysteine crosslinking Step 2: a) Determine ATPase activity / nucleotide occlusion and b) the physiological relevance of arrested state Step 3: Structural studies

  9. Results Step 1 Arrest ABCE1 in different conformations by cysteine crosslinking Goal: • 100% crosslinking • No over-oxidation to sulfoxides

  10. Overview: SulfolobussolfataricusABCE1 variants K84 Y-loop variant D459 N108 Walker A variant S461 Q-loop variant G462 Y168 S488 D-loop variant S241 ATP-site 2 variant N204 E538 HadasLeonov, Bert de Groot and Helmut Grubmüller, MPI of Biophysical Chemistry, Göttingen

  11. Results Step 2 Probe arrested conformation by 30S binding Goal: • Oxidative cysteinecrosslinkingwithFeSdomain • Sucrose density centrifugation with S. solfataricuslysate

  12. 30S binding: Sucrosedensitycentrifugation FeS lysate ABCE1 (nucleotide) 10% top centrifugation 30S 50S 30% Griffithset. al. 2000 top 30S kDa c 70 - AMPPNP ssABCE1_wt: 0.5 μM protein 2 mMnucleotide c 70 - ADP c = ssABCE1_wt/ssABCE1_∆FeS α -His (1:2000)

  13. D-loop variant – 30S binding(SDG) FeS FeS 30S ox kDa c 70 - AMPPNP red OX 70 - c ADP D-loop variant c c 70 - OX ADP c = ssABCE1_wt/ssABCE1_∆FeS α -His (1:2000) wildtype

  14. Summary Protein biosynthesis… • … is a cyclic process • Termination and initiation are connected by ribosome recycling • … is an unusual member of the ABC protein family • … recycles ribosomes in eukaryotes and archaea • Dynamic mechanism is not yet understood but can hopefully be accessed (soon)by cysteine-cysteine crosslinking ABCE1…

  15. ThankYou • Kristin Kiosze, Milan Gerovac & Lab1.21 • Dr. Umar Jan • Prof. Robert Tampé • All colleagues

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