Elongation factors on the ribosome. Curr Opin Struct Biol

University of Aarhus, Department of Molecular Biology, Gustav Wieds Vej 10C, 8000 Arhus C, Denmark.
Current Opinion in Structural Biology (Impact Factor: 7.2). 07/2005; 15(3):349-54. DOI: 10.1016/
Source: PubMed


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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    • "Similar dissociation mechanism is exploited by the elongation factor Tu (dubbed EF1A in Archaea and Eukarya), which is a GTPase that positions aminoacyl-tRNA complexes within the A-site of prokaryotic ribosomes. Once the aminoacyl-tRNA complex is positioned in the ribosome via codon–anticodon base-pairing, EF-Tu undergoes a conformational change powered by GTP hydrolysis that results in the dissociation of aminoacyl-tRNA from EF-Tu (51). The bacterial DEZ complex, on the other hand, does not co-purify with tRNA and it shows low binding affinity for tRNA in vitro, indicating formation of unstable nucleoprotein complexes (not shown, unpublished data). "
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    ABSTRACT: N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.
    Nucleic Acids Research 08/2013; 41(20). DOI:10.1093/nar/gkt720 · 9.11 Impact Factor
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    • "During elongation, the ribosome repeats the cycle of selecting a transfer RNA (tRNA) molecule matching the codon in the 30S A site, incorporating the amino acid from the selected A-site tRNA into the polypeptide on the P-site tRNA, translocating the A-and P-site tRNAs to the P and E sites, and stepping precisely three bases in the 3 0 direction (Korostelev et al., 2008; Wintermeyer et al., 2004; Zaher and Green, 2009). To elongate with an optimal balance of speed and accuracy, the ribosome employs G protein elongation factors (elongation factor thermo unstable [EF-Tu] and elongation factor G [EF-G] in bacteria) to facilitate key steps during the process (Nilsson and Nissen, 2005). Using the energy from guanosine triphosphate (GTP) hydrolysis, EF-Tu enhances the rate and specificity of tRNA selection and EF-G catalyzes translocation. "
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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 · 8.36 Impact Factor
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    • "Indeed, some surface proteins are assumed to be attached to the cell wall in a non-covalent way and have been reported to be lost during mild standard manipulations [26,27]. EF-Tu(elongation factor thermo unstable) was identified as a cell wall related protein in this study, which was also been found as cell wall protein in other studies [28]. Translation elongation factors are responsible for two main processes during protein synthesis on the ribosome [29]. "
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    ABSTRACT: The usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis. A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. smegmatis. An enzymatic cell surface shaving method was used to determine the surface-exposed proteins. As a result, a total of 390 cell wall proteins and 63 surface-exposed proteins were identified. Further analysis of the 390 cell wall proteins provided the theoretical molecular mass and pI distributions and determined that 26 proteins are shared with the surface-exposed proteome. Detailed information about functional classification, signal peptides and number of transmembrane domains are given next to discussing the identified transcriptional regulators, transport proteins and the proteins involved in lipid metabolism and cell division. In short, a comprehensive profile of the M. smegmatis cell wall subproteome is reported. The current research may help the identification of some valuable vaccine and drug target candidates and provide foundation for the future design of preventive, diagnostic, and therapeutic strategies against mycobacterial diseases.
    BMC Microbiology 04/2010; 10(1):121. DOI:10.1186/1471-2180-10-121 · 2.73 Impact Factor
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