Elongation factor ternary complex

The now deacylated tRNA is attached by its anticodon to the P site at one end and by the open GGA tail to an exit (E) site on the large ribosomal subunit (Figure 38-8). At this point, elongation factor 2 (EE2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EFlA-GTP and another cycle of elongation. 

The same method has been applied to measure the Eli domain orientation when the protein is in complex with its RNA parmer or both RNA and thiostrepton antibiotic. The additional RDCs revealed a rearrangement of the N-terminal domain of Ell placing it closer to the RNA after binding of thiostrepton. HADDOCK has been used to calculate a model of the ternary stmcture of the Ell protein in complex with RNA and antibiotic. Based on the orientational data, the dynamics and the docking model, it seems that thiostrepton locks the domain conformation of Ell in a rigid (inhibitory) state. The antibiotic thiostrepton interferes with the interaction of elongation factors to this Ell-RNA region, which has a dramatic effect on the level of protein synthesis by the ribosome. 

Figure 4 Minimal mechanism by which aa-RNs are incorporated into nascent protein (see discussion for comments). AatRNA, aminoacyl tRNA EFTu, elongation factor GTP, guanosine triphosphate GDP, guanosine diphosphate R, ribosome T, ternary complex )aa-tRNA GTP EFTu). Redrawn from Thompson 198831...

The tRNA selection pathway and kinetic proofreading. Aminoacyl-tRNA is delivered to the ribosomal A-site as a ternary complex with elongation factor EF-Tu and GTP. The events between the initial binding of the ternary complex and the incorporation of the amino acid into the peptide have been dissected into several kineti-cally distinguishable steps, as shown in Figure 13.17. 

Studies on the complex between the elongation factor EF-Tu. GTP (M 46,000 Rq 2.5 nm) with aminoacylated tRNA (nucleotides 24,500) benefit from the closer similarity in size of the two components [379-381]. X-ray titrations show that a 1 1 complex is formed, and the Rq of 3.6 nm and the distance distribution functions suggested that an extended complex is formed [380]. Subsequent neutron studies have, however, proposed that EF-Tu exists in a monomer-dimer equilibrium. The Rq values of 2.0 nm for monomeric EF-Tu, 2.3 nm for aminoacylated tRNA, and 2.6 nm for the ternary complex have led to the alternative proposition of a compact structure for this complex [381]. 

By the above criteria, both analogs form a specific ternary complex with the ribosomes and elongation factor G that is close in efficiency to the native GTP complex. In particular, the ribose photoanalog of GTP is a substrate of the GTPase reaction to test this activity it was synthesized from [y- -P]GTP. Some characteristics of the y-phosphate analog of GTP (the absence of hydrolysis by GTPase, noneffectivity of... 

The preparation of these photoanalogs of GTP is simple and can be used practically without modification for the radioactive microsynthesis of other photoactivated or chemically specific affinity analogs of GTP. It cannot be concluded, however, that the several groups attached to the nucleotide may disturb its function. In our case, for example, a GTP derivative similar in structure to the y-(4-azidobenzyl) amide of GTP, but containing an aromatic amine, the y-(4-azido) anilide of GTP, is almost without inhibitory ability for the cell-free poly (U)-dependent synthesis of polyphenylalanine and does not form a ternary complex with ribosomes and elongation factor G. 

After dissociation of the three initiation factors, the 70S initiation complex is ready to bind a ternary complex consisting of aminoacyl-tRNA, elongation factor EF-Xu and GXP. This complex binds to the ribosomal acceptor-site, the A-site, in such a way that the anticodon of the tRNA is in close contact with the complementary codon on the... 

The cycle of EF-Tu during protein biosynthesis, emerging from the studies on T. thermophilus system [2] conducted in our laboratory, is shown in Fig. 19.10. EF-Tu GDP binds EF-Ts and a tetrameric complex (EF-Tu EF-Ts)2 is formed. The dissociation constant for this interaction is about 10 M, in the case of T. thermophilus elongation factors. The functional reason for the formation of an (EF-Tu EF-Ts)2 tetramer, also observed with other elongation factors, is not yet understood. EF-Ts functions as nucleotide exchange factor for EF-Tu since it promotes dissociation of GDP. Aminoacyl-tRNA in the presence of GTP is required for dissociation of T. thermophilus (EF-Tu EF-Ts)2 and aminoacyl-tRNA EF-Tu GTP ternary complex is formed. The high concentration of GTP (10 M) and aminoacyl-tRNA (10 " M) in the bacterial cell is the main factor which drives the cycle of EF-Tu... 

Addition of each incoming amino acid requires the cooperation of three elongation factors. Elongation factor Tu (EF-Tu) is the most abundant protein in E. coli, with about 100,000 copies per cell, or 5% of the cell s protein. This protein is a GTPase, and the EF-Tu GTP complex specifically binds aminoacyl-tRNAs (AA-tRNAs). Formation of the ternary complex (EF-Tu GTP AA-tRNA) protects the ester bond (linking the amino acid to its cognate tRNA) from hydrolysis, and transports the AA-tRNA to the ribosomal A-site. Once the correct codon-anticodon interaction is confirmed, ribosome-triggered hydrolysis of... 

The crystal structure of the ternary complex with Phe-tRNA (EF-Tu GDPNP Phe-tRNA ) demonstrated that the EF-Tu structure in the ternary complex is similar to that in the EF-Tu GDPNP structure. Thus, binding of AA-tRNA does not alter the EF-Tu conformation. The ternary complex is quite elongated (Fig. 10), with the tRNA anticodon pointing away from EF-Tu, and close contacts are observed only between the factor and the T-stem, 3 -CCA-AA, and 5 -phosphate of the AA-tRNA. The Phe-tRNA structure is also not significantly altered upon binding to EF-Tu. 

FIGURE 10 Comparison of elongation factor structures. The crystal structures of the EF-Tu GDPNP tRNA ternary complex (left) and EF-G (right) revealed that Domains 3, 4, and 5 of EF-G mimic the conformation of EF-Tu-bound tRNA. Several other translational factors have been determined or predicted to similarly mimic the tRNA structure. 

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