Modification, inhibition and function of elongation factor EF-P
Final Report Abstract
Protein synthesis in the cell occurs on macromolecular machines called ribosomes. The ribosome synthesizes polypeptide chains by providing a platform upon which peptide-bond formation can occur between a peptidyl-tRNA located at the ribosomal P-site and an amino acyl-tRNA in the A-site. However, the ribosome cannot form peptide bonds between all amino acids with the same efficiency; this is exemplified by the amino acid proline. Proline, which has an imino group instead of a primary amino group in other amino acids, has been shown to be a particularly poor substrate for peptide-bond formation, both as a donor in the P-site and as an acceptor in the A-site. In fact, ribosomes stall when attempting to incorporate three or more consecutive proline residues (PPP) into the polypeptide chain. This translational arrest due to polyproline stretches is relieved by the translation elongation factor EF-P in bacteria and a/eIF-5A in archaea/eukaryotes, however, a structural basis for this rescue has been lacking. Here we have determined cryo-EM structures of polyproline-stalled ribosomes in the presence and absence of EF-P, as well as eIF-5A in complex with the yeast 80S ribosome. Our structures suggest that stalling due to polyproline-stretches is due to destabilization of the P-site tRNA, which can even induce drop-off for short peptidyltRNAs. By contrast, the presence of EF-P or a/eIF-5A interacts and stabilizes the P- tRNA and positions the CCA-end with an optimal geometry for peptide bond formation. We identified rhamnoslyation as the modification present in EF-P proteins from γ-proteobacteria, such as Pseudomonas aeruginosa and Shewanella oneidensis. We characterized and structurally identified a new subfamily of EF-P in the phylum Actinobacteria, which includes the medically and economically important genera Corynebacterium, Mycobacterium and Streptomyces. This project paves the way for the development of tailored designer EF-Ps for various applications.
Publications
- (2020) Structure and Function of an Elongation Factor P Subfamily in Actinobacteria. Cell reports 30 (13) 4332-4342.e5
Pinheiro, Bruno; Scheidler, Christopher M.; Kielkowski, Pavel; Schmid, Marina; Forné, Ignasi; Ye, Suhui; Reiling, Norbert; Takano, Eriko; Imhof, Axel; Sieber, Stephan A.; Schneider, Sabine; Jung, Kirsten
(See online at https://doi.org/10.1016/j.celrep.2020.03.009) - Argininerhamnosylation as new strategy to activate translation elongation factor P. Nature Chem Biol. 2015 Apr;11(4):266-70
Lassak J, Keilhauer EC, Fürst M, Wuichet K, Gödeke J, Starosta AL, Chen JM, Søgaard-Andersen L, Rohr J, Wilson DN, Häussler S, Mann M, Jung K
(See online at https://doi.org/10.1038/NCHEMBIO.1751) - Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea. Archaea. 2016 Dec 8: 7316725
Prunetti L, Graf M, Blaby IK, Peil L, Makkay AM, Starosta AL, Papke RT, Oshima T, Wilson DN, de Crécy-Lagard V
(See online at https://doi.org/10.1155/2016/7316725) - Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A. Mol Microbiol. 2016 Jan;99(2):219-35
Lassak J, Wilson DN, Jung K
(See online at https://doi.org/10.1111/mmi.13233) - Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome. Nucleic Acids Res. 2016 Feb 29;44(4):1944-51
Schmidt C, Becker T, Heuer A, Braunger K, Shanmuganathan V, Pech M, Berninghausen O, Wilson DN, Beckmann R
(See online at https://doi.org/10.1093/nar/gkv1517) - Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF- P. Mol Cell. 2017 Nov 2;68(3):515-527
Huter P, Arenz S, Bock LV, Graf M, Frister JO, Heuer A, Peil L, Starosta AL, Wohlgemuth I, Peske F, Nováček J, Berninghausen O, Grubmüller H, Tenson T, Beckmann R, Rodnina MV, Vaiana AC, Wilson DN
(See online at https://doi.org/10.1016/j.molcel.2017.10.014) - Switching the post- translational modification of translation elongation factor EF-P. Front Microbiol. 2019 10:1148
Volkwein W, Krafczyk R, Kumar PAJ, Parr M, Mankina E, Macošek J, Guo Z, Fürst MJLJ, Pfab M, Frishman D, Hennig J, Jung K, Lassak J
(See online at https://doi.org/10.3389/fmicb.2019.01148)
