Dynamics in Protein Synthesis, Structural Studies of Translation Factors

Abstract: 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.