Ribosome rearrangements during translocation
Final Report Abstract
The ribosome is a macromolecular machine consisting of RNA and proteins that synthesizes proteins in all cells. Amino acids are carried to the ribosome bound to tRNAs in activated form and polymerized in the sequence encoded in the base sequence ofthe mRNA, which is a copy of the respective gene. Three bases each (out of four) form a codon that encodes one particular amino acid (out of 20 standard amino acids). The three main phases of protein synthesis are initiation, elongation, and termination. During these phases, different sets of accessory factors bind to the ribosome and determine the respective function of the ribosome. In this project, we have concentrated on one step of the elongation cycle, the translocation ofthe tRNAs that follows the formation of the peptide bond. During translocation, two tRNAs move by distances of 20 Ä or more to adjacent sites on the ribosome. The movement of the tRNAs is coupled to the movement of the mRNA by one codon. In bacteria, translocation is promoted by elongation factor G (EF-G), a large GTPase consisting of five domains, which hydrolyzes GTP during the process. The project was started when we showed - contrary to the then general belief - that GTP hydrolysis takes place prior to, and drives, tRNA movement. We have carried out an extensive kinetic analysis that revealed that ribosome-induced GTP hydrolysis by EF-G results in a coupled conformational rearrangement of EF-G and the ribosome ("unlocking") that is rate-limiting for tRNA-mRNA movement. The inorganic phosphate (Pi) resulting from GTP hydrolysis is kept within the nucleotide binding pocket, which closes upon EF-G binding to the ribosome, and released only after unlocking to allow the dissociation of EF-G from the ribosome. As revealed by cryo-electron microscopic analysis, the spontaneous movement ofthe tRNAs is weakly coupled to conformational fluctuations of the ribosome. The role of EF-G is three-fold: (i) EF-G-GTP binds to, and stabilizes, the ratcheted conformation of the ribosome; (ii) upon GTP hydrolysis it accelerates the formation of the "unlocked" state of the ribosome, thereby allowing for rapid movement of tRNAs; (iii) it functions as a Brownian ratchet by blocking the A site for backward movement ofthe tRNAs, biasing tRNA movement into the forward direction.
Publications
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Rodnina, M.V., Savelsbergh, A., Katunin, V.l., and Wintermeyer, W.
- (1998) Form follows function: Structure of an elongation factor G-ribosome complex. Proc. Natl. Acad. Sci. 95, 7237-7239 (Commentary)
Rodnina, M. V., Wintermeyer, W.
- (1999) Dynamics of translation on the ribosome: molecular mechanics of translocation. FEMS Microbiology Reviews 23, 317-333
Rodnina, M. V., Savelsbergh, A., Wintermeyer, W.
- (1999). Thiostrepton inhibits the tumover but not the GTPase of elongation factor G on the ribosome. Proc. Natl. Acad. Sci. USA 96, 9586-9590
Rodnina, M.V., Savelsbergh, A., Matassova, N.B., Katunin, V.l., Semenkov, Y.P., and Wintermeyer, W.
- (2000) GTPase mechanisms and functions of translation factors on the ribosome. Biol. Chem. 381, 377-387
Rodnina, M. V., Stark, H., Savelsbergh, A., Wieden, H. J., Mohr, D., Matassova, N. B., Peske, F-, Daviter, T., Gualerzi, C, Wintermeyer, W.
- (2000) Mechanisms of partial reactions of the elongation cycle catalyzed by elongation factors Tu and G- In: The Ribosome: Structure, Funcfion, Antibiotics, and Cellular Interactions. Eds. Garrett, R. A., Douthwaite, S. R., Liljas, A., Matheson, A. T., Moore, P. B., Noller, H. F. American Society of Microbiology Press, Washington D.C, pp. 301-317
Rodnina, M. V., Pape, T., Savelsbergh, A., Mohr, D., Matassova, N. B., Wintermeyer, W.
- (2000) Translational elongation factor G: a GTP-driven motor of the ribosome. In: Essays in Biochemistry 35, Molecular Motors, pp. 117-130
Wintermeyer, W., Rodnina, M.V.
- (2000). Arginines 29 and 59 of elongation factor G are important for GTP hydrolysis or translocation on the ribosome. EMBO J. 19, 3458-3464
Mohr, D., Wintermeyer, W., and Rodnina, M.V.
- (2000). Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome. Mol. Cell 6, 501-505
Peske, F., Matassova, N.B., Savelsbergh, A., Rodnina, M.V., and Wintermeyer, W.
- (2000). Energetic contribution of tRNA hybrid state formation to translocation catalysis on the ribosome. Nat. Struct. Biol. 7, 1027-1031
Semenkov, Y.P., Rodnina, M.V., and Wintermeyer, W.
- (2000). Largescale movement of elongation factor G and extensive conformational change of the ribosome during translocation. Cell 100, 301-309
Stark, H., Rodnina, M.V., Wieden, H.J., van Heel, M., and Wintermeyer, W.
- (2000). Role of domains 4 and 5 in elongation factor G functions on the ribosome. J. Mol. Biol. 300, 951 -961
Savelsbergh, A., Matassova, N.B., Rodnina, M.V., and Wintermeyer, W.
- (2000). Stimulation of the GTPase activity of translation elongation factor G by ribosomal protein L7/12. J. Biol. Chem. 275, 890-894
Savelsbergh, A., Mohr, D., Wilden, B., Wintermeyer, W., and Rodnina, M.V.
- (2001) Mechanism of elongation factor G function in tRNA translocation on the ribosome. Cold Spring Harbor Symposia on Quantitative Biology. Vol. 66, 449-457
Wintermeyer, W., Savelsbergh, A., Semenkov, Y. P., Katunin, V. I., Rodnina, M. V.
- (2001) Mechanism of tRNA translocation on the ribosome. Molecular Biology (Moscow) 35, 559-568
Rodnina, M. V., Semenkov, Y. P., Savelsbergh, A., Katunin, V. I., Peske, F., Wilden, B., Wintermeyer, W.
- (2001). Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome. RNA 7, 1879-1885
Matassova, A.B., Rodnina, M.V., and Wintermeyer, W.
- (2002). GTPase activation of elongafion factors Tu and G on the ribosome. Biochemistry 41, 12520-12528
Mohr, D., Wintermeyer, W., and Rodnina, M.V.
- (2003) Das Ribosom: Struktur und Funktionen eines Mega-Ribozyms. Biospektrum 9, 138-141
Rodnina, M. V., Wintermeyer, W.
- (2003). An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation. Mol. Cell 11, 1517-1523
Savelsbergh, A., Katunin, V.l., Mohr, D., Peske, F., Rodnina, M.V., and Wintermeyer, W.
- (2004) Mechanisms of elongation on the ribosome: dynamics ofa macromolecular machine. Biochem. Soc. Trans. 32, 733-737
Wintermeyer, W., Peske, F., Beringer, M., Gromadski, K. B., Savelsbergh, A., und Rodnina, M. V.
- (2004). Conformarional changes of the small ribosomal subunit during elongafion factor G-dependent tRNA-mRNA translocation. J. Mol. Biol. 343, 1183-1194
Peske, F., Savelsbergh, A., Katunin, V.l., Rodnina, M.V., and Wintermeyer, W.
- (2005). Control of phosphate release from elongation factor G by ribosomal protein L7/I2. EMBOJ. 24,4316- 4323
Savelsbergh, A., Mohr, D., Kothe, U., Wintermeyer, W., and Rodnina, M.V.
- (2005). Sequence of steps in ribosome recycling as defined by kineric analysis. Mol. Cell 18, 403-412
Peske, F., Rodnina, M.V., and Wintermeyer, W.
- (2006) Colicins and their potenfial in cancer treatment. Blood Cells, Molecules and Diseases 38, 15-18
Lancaster, L. E., Wintermeyer, W., Rodnina, M. V.
- (2006). Involvement of helix 34 of 16 S rRNA in decoding and translocation on the ribosome. J. Biol. Chem. 281,35235-35244
Kubarenko, A., Sergiev, P., Wintermeyer, W., Dontsova, 0., and Rodnina, M.V.
- (2006). Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome. Proc. Natl. Acad. Sci. USA 103, 13670-13675
Wilden, B., Savelsbergh, A., Rodnina, M.V., and Wintermeyer, W.
- (2007). Spontaneous reverse movement of mRNA-bound tRNA through the ribosome. Nat. Struct Mol. Biol, 14,318-324
Konevega, A,L., Fischer, N., Semenkov, Y.P., Stark, H., Wintermeyer, W., and Rodnina, M.V.
- (2008). Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Eschenchia coli ribosomes. Mol. Microbiol 69, 390-401
Lancaster, L.E., Savelsbergh, A., Kleanthous, C, Wintermeyer, W., and Rodnina, M.V.
- (2008). Structure of ratcheted ribosomes with tRNAs in hybrid states. Proc. Nafl. Acad. Sci. USA 105, 16924-16927
Julian, P., Konevega, A.L., Scheres, S.H., Lazaro, M., Gil, D., Wintermeyer, W., Rodnina, M.V., and Valle, M.
- (2009) Recent mechanistic insight into eukaryofic ribosomes. Curr. Opin. Cell Biol. 21, 435-443
Rodnina, M.V., Wintermeyer, W.
- (2009). Distinct functions of elongation factor G in ribosome recycling and translocation. RNA 15, 772-780
Savelsbergh, A., Rodnina, M.V., and Winlermeyer, W.
