EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes.[2][3][4] It is found in eukaryotic mitochondria as TUFM.[5]
Elongation Factor Thermo Unstable
EF-Tu (blue) complexed with tRNA (red) and GTP (yellow) [1]
As a family of elongation factors, EF-Tu also includes its eukaryotic and archaeal homolog, the alpha subunit of eEF-1 (EF-1A).
Backgroundedit
Elongation factors are part of the mechanism that synthesizes new proteins through translation in the ribosome. Transfer RNAs (tRNAs) carry the individual amino acids that become integrated into a protein sequence, and have an anticodon for the specific amino acid that they are charged with. Messenger RNA (mRNA) carries the genetic information that encodes the primary structure of a protein, and contains codons that code for each amino acid. The ribosome creates the protein chain by following the mRNA code and integrating the amino acid of an aminoacyl-tRNA (also known as a charged tRNA) to the growing polypeptide chain.[6][7]
There are three sites on the ribosome for tRNA binding. These are the aminoacyl/acceptor site (abbreviated A), the peptidyl site (abbreviated P), and the exit site (abbreviated E). The P-site holds the tRNA connected to the polypeptide chain being synthesized, and the A-site is the binding site for a charged tRNA with an anticodon complementary to the mRNA codon associated with the site. After binding of a charged tRNA to the A-site, a peptide bond is formed between the growing polypeptide chain on the P-site tRNA and the amino acid of the A-site tRNA, and the entire polypeptide is transferred from the P-site tRNA to the A-site tRNA. Then, in a process catalyzed by the prokaryotic elongation factor EF-G (historically known as translocase), the coordinated translocation of the tRNAs and mRNA occurs, with the P-site tRNA moving to the E-site, where it dissociates from the ribosome, and the A-site tRNA moves to take its place in the P-site.[6][7]
Biological functionsedit
Protein synthesisedit
EF-Tu participates in the polypeptide elongation process of protein synthesis. In prokaryotes, the primary function of EF-Tu is to transport the correct aa-tRNA to the A-site of the ribosome. As a G-protein, it uses GTP to facilitate its function. Outside of the ribosome, EF-Tu complexed with GTP (EF-Tu • GTP) complexes with aa-tRNA to form a stable EF-Tu • GTP • aa-tRNA ternary complex.[8] EF-Tu • GTP binds all correctly-charged aa-tRNAs with approximately identical affinity, except those charged with initiation residues and selenocysteine.[9][10] This can be accomplished because although different amino acid residues have varying side-chain properties, the tRNAs associated with those residues have varying structures to compensate for differences in side-chain binding affinities.[11][12]
The binding of an aa-tRNA to EF-Tu • GTP allows for the ternary complex to be translocated to the A-site of an active ribosome, in which the anticodon of the tRNA binds to the codon of the mRNA. If the correct anticodon binds to the mRNA codon, the ribosome changes configuration and alters the geometry of the GTPase domain of EF-Tu, resulting in the hydrolysis of the GTP associated with the EF-Tu to GDP and Pi. As such, the ribosome functions as a GTPase-activating protein (GAP) for EF-Tu. Upon GTP hydrolysis, the conformation of EF-Tu changes drastically and dissociates from the aa-tRNA and ribosome complex.[4][13] The aa-tRNA then fully enters the A-site, where its amino acid is brought near the P-site's polypeptide and the ribosome catalyzes the covalent transfer of the polypeptide onto the amino acid.[10]
In the cytoplasm, the deactivated EF-Tu • GDP is acted on by the prokaryotic elongation factor EF-Ts, which causes EF-Tu to release its bound GDP. Upon dissociation of EF-Ts, EF-Tu is able to complex with a GTP due to the 5– to 10–fold higher concentration of GTP than GDP in the cytoplasm, resulting in reactivated EF-Tu • GTP, which can then associate with another aa-tRNA.[8][13]
Maintaining translational accuracyedit
EF-Tu contributes to translational accuracy in three ways. In translation, a fundamental problem is that near-cognate anticodons have similar binding affinity to a codon as cognate anticodons, such that anticodon-codon binding in the ribosome alone is not sufficient to maintain high translational fidelity. This is addressed by the ribosome not activating the GTPase activity of EF-Tu if the tRNA in the ribosome's A-site does not match the mRNA codon, thus preferentially increasing the likelihood for the incorrect tRNA to leave the ribosome.[14] Additionally, regardless of tRNA matching, EF-Tu also induces a delay after freeing itself from the aa-tRNA, before the aa-tRNA fully enters the A-site (a process called accommodation). This delay period is a second opportunity for incorrectly charged aa-tRNAs to move out of the A-site before the incorrect amino acid is irreversibly added to the polypeptide chain.[15][16] A third mechanism is the less well understood function of EF-Tu to crudely check aa-tRNA associations and reject complexes where the amino acid is not bound to the correct tRNA coding for it.[11]
Other functionsedit
EF-Tu has been found in large quantities in the cytoskeletons of bacteria, co-localizing underneath the cell membrane with MreB, a cytoskeletal element that maintains cell shape.[17][18] Defects in EF-Tu have been shown to result in defects in bacterial morphology.[19] Additionally, EF-Tu has displayed some chaperone-like characteristics, with some experimental evidence suggesting that it promotes the refolding of a number of denatured proteins in vitro.[20][21]
The GTP-binding domain I undergoes a dramatic conformational change upon GTP hydrolysis to GDP, allowing EF-Tu to dissociate from aa-tRNA and leave the ribosome.[27] Reactivation of EF-Tu is achieved by GTP binding in the cytoplasm, which leads to a significant conformational change that reactivates the tRNA-binding site of EF-Tu. In particular, GTP binding to EF-Tu results in a ~90° rotation of domain I relative to domains II and III, exposing the residues of the tRNA-binding active site.[28]
Domain 2 adopts a beta-barrel structure, and is involved in binding to charged tRNA.[29] This domain is structurally related to the C-terminal domain of EF2, to which it displays weak sequence similarity. This domain is also found in other proteins such as translation initiation factor IF-2 and tetracycline-resistance proteins. Domain 3 represents the C-terminal domain, which adopts a beta-barrel structure, and is involved in binding to both charged tRNA and to EF1B (or EF-Ts).[30]
Along with the ribosome, EF-Tu is one of the most important targets for antibiotic-mediated inhibition of translation.[8] Antibiotics targeting EF-Tu can be categorized into one of two groups, depending on the mechanism of action, and one of four structural families. The first group includes the antibiotics pulvomycin and GE2270A, and inhibits the formation of the ternary complex.[36] The second group includes the antibiotics kirromycin and enacyloxin, and prevents the release of EF-Tu from the ribosome after GTP hydrolysis.[37][38][39]
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^ abLaRiviere FJ, Wolfson AD, Uhlenbeck OC (October 2001). "Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation". Science. 294 (5540): 165–8. Bibcode:2001Sci...294..165L. doi:10.1126/science.1064242. PMID 11588263. S2CID 26192336.
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^Whitford PC, Geggier P, Altman RB, Blanchard SC, Onuchic JN, Sanbonmatsu KY (June 2010). "Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways". RNA. 16 (6): 1196–204. doi:10.1261/rna.2035410. PMC2874171. PMID 20427512.
^Noel JK, Whitford PC (October 2016). "How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome". Nature Communications. 7: 13314. Bibcode:2016NatCo...713314N. doi:10.1038/ncomms13314. PMC5095583. PMID 27796304.
^Defeu Soufo HJ, Reimold C, Linne U, Knust T, Gescher J, Graumann PL (February 2010). "Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein". Proceedings of the National Academy of Sciences of the United States of America. 107 (7): 3163–8. Bibcode:2010PNAS..107.3163D. doi:10.1073/pnas.0911979107. PMC2840354. PMID 20133608.
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^Mayer F (2006-01-01). "Cytoskeletal elements in bacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli as revealed by electron microscopy". Journal of Molecular Microbiology and Biotechnology. 11 (3–5): 228–43. doi:10.1159/000094057. PMID 16983198. S2CID 23701662.
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^Kudlicki W, Coffman A, Kramer G, Hardesty B (December 1997). "Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing". The Journal of Biological Chemistry. 272 (51): 32206–10. doi:10.1074/jbc.272.51.32206. PMID 9405422.
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^Wiborg O, Andersen C, Knudsen CR, Clark BF, Nyborg J (August 1996). "Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA". The Journal of Biological Chemistry. 271 (34): 20406–11. doi:10.1074/jbc.271.34.20406. PMID 8702777.
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^Ann DK, Moutsatsos IK, Nakamura T, Lin HH, Mao PL, Lee MJ, Chin S, Liem RK, Wang E (June 1991). "Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin". J. Biol. Chem. 266 (16): 10429–37. doi:10.1016/S0021-9258(18)99243-4. PMID 1709933.
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^Selva E, Beretta G, Montanini N, Saddler GS, Gastaldo L, Ferrari P, Lorenzetti R, Landini P, Ripamonti F, Goldstein BP (July 1991). "Antibiotic GE2270 a: a novel inhibitor of bacterial protein synthesis. I. Isolation and characterization". The Journal of Antibiotics. 44 (7): 693–701. doi:10.7164/antibiotics.44.693. PMID 1908853.
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External linksedit
Peptide+Elongation+Factor+Tu at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
Overview of all the structural information available in the PDB for UniProt: P49410 (Elongation factor Tu, mitochondrial) at the PDBe-KB.