Elongation factors on the ribosome.
ABSTRACT The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.
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ABSTRACT: Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition.Cell Reports 02/2013; 3(2):497-508. DOI:10.1016/j.celrep.2013.01.027 · 7.21 Impact Factor
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ABSTRACT: The ribosome is a ribozyme whose active site, the peptidyl trans-ferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A- > P-site passage of the tRNA terminus in the peptidyl-transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC the rotatory motion leads to stereochemistry suitable for peptide bond formation as well as for substrate mediated catalysis, consistent with quantum mechanical calculations illuminating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant of PTC mobility and supported the hypothesis that the ancient ribosome produced single peptides bonds and non-coded chains, utilizing nucleotide conjugated amino acids. Genetic control of the reaction evolved after polypeptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotides orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures. The increase in antibiotic resistance among pathogenic bacterial strains poses a significant health threat. Therefore, improvement of existing antibiotics and the design of advance drugs are urgently needed. Ribosomes provide binding sited for many antibiotic families, utilizing their inherent functional flexibility, which triggers induced fit mechanism by remote interactions, and facilitates antibiotics synergism as well as reshaping less suitable binding pockets, leading to clinical usefulness even for antibiotics that bind to conserved functional regions. Exploitation of the diverse properties of antibiotics binding and benefiting from the detailed structural information that keeps emerging, should result in significant antibiotics improvement.05/2009: pages 121-155;
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ABSTRACT: The D60A mutant of the elongation factor (EF) 1alpha from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1alpha; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.Biochimie 05/2009; 91(7):835-42. DOI:10.1016/j.biochi.2009.04.003 · 3.12 Impact Factor