Dynamics in Protein Synthesis, Structural Studies of Translation Factors
Research output: Thesis › Doctoral Thesis (compilation)
Protein biosynthesis is performed on ribosomes. The ribosome is a complex of ribosomal RNA and proteins forming two subunits. A messenger RNA strand is decoded on the small subunit by charged aminoacyl transfer RNA (aa-tRNA) molecules and each tRNA molecule delivers its amino acid to the growing polypeptide chain on the large subunit. RNA performs the central reactions and ribosomal proteins and factors assist in the process. The elongation factors EF-Tu and EF-G are GTPases alternately binding to the ribosomal factor-binding site. EF-Tu inserts an aa-tRNA molecule in the ribosomal tRNA acceptor site (A-site) and EF-G catalyzes the synchronized translocation of tRNAs and the messenger between ribosomal sites. The crystal structure of EF-G carrying the His573Ala mutation has been determined and it revealed the structure of domain III of EF-G. This domain extends the tRNA L-shape mimic recurrently observed among translation factors to include the tRNA acceptor stem. An observed rotation of domains III, IV and V versus domains I and II brings domains III and I in close contact. Mutations conferring strong resistance towards the antibiotic fusidic acid (FA) have been identified to flank this domains interface and a possible FA binding site has been identified. Impairment by FA after GTP hydrolysis and structural rearrangements of EF-G have been proposed to prevent the factor from dissociating after translocation and thereby block protein synthesis. The crystal structure of a FA hypersensitive mutant with an increased affinity for GTP supports this view. During acute amino acid starvation the ribosome associated stringent factor RelA catalyses the formation of pppGpp from ATP and GTP as a response to an elevated frequency of uncharged tRNAs in the ribosomal A-site. The stringent response factor, GPP, hydrolyzes pppGpp to the active ppGpp affecting levels of transcription, protein synthesis and protein degradation. GPP has successfully been crystallized and structure determination is currently under way.The elongation factor P catalyzing formation of the first peptide bond of the nascent peptide chain on the ribosome has been crystallized and structure determination is in progress.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Award date||2002 Mar 8|
|Publication status||Published - 2002|
Defence details Date: 2002-03-08 Time: 13:15 Place: Kemicentrum, Sölvegatan 39, hörsal A External reviewer(s) Name: Nyborg, Jens Title: Prof Affiliation: [unknown] --- Article: I. Laurberg, M., Kristensen, O., Martemyanov, K., Gudkov, A. T. & Liljas, A. Crystal Structure of a Fusidic Acid hypersensitive mutant of Elongation Factor G, G16V. – Submitted to Journal of Biological Chemistry. Article: II. Laurberg, M., Kristensen, O., Su, XD. & Liljas, A. New crystal form of Thermus thermophilus elongation factor G indicates crystallographic limitations imposed on molecular flexibility. – Submitted to FEBS Letters. Article: III. Kristensen, O., Laurberg, M. & Gajhede, M. Crystallization of a stringent response factor from Aquifex aeolicus. – Submitted to Acta Crystallographica Section D. Article: IV. Kristensen, O. & Laurberg, M. (2002). Expression, refolding and crystallization of Aquifex aeolicus elongation factor P. - Submitted to Acta Crystallographica Section D. Article: V. Laurberg, M., Kristensen, O., Martemyanov, K., Gudkov, A. T., Nagaev, I., Hughes, D. & Liljas, A. (2000). Structure of a Mutant EF-G Reveals Domain III and Possibly the Fusidic Acid Binding Site. J Mol Biol 303, 593-603. Article: VI. Liljas, A., Kristensen, O., Laurberg, M., Al-Karadaghi, S., Gudkov, A., Martemyanov, K., Hughes, D. & Nagaev, I. (1999). The states, conformational dynamics and fusidic acid resistant mutants of EF-G. In The Ribosome: Structure, Function, Antibiotics and Cellular Interactions, pp. 359-365. Edited by R. A. Garrett, S. R. Douthwaite, A. Liljas, A. T. Matheson, P. B. Moore & H. F. Noller. Washington, D. C.: 2000 AMS Press.