Aminoacyl-tRNA in complex with EF-Tu GTP

For codon-specific binding of aminoacyl-tRNA to the ribosome the tRNA must form a complex with the elongation factor Tu. The complex formation manifests itself also in the imino resonance region of the NMR spectrum of the tRNA. Since the molecular mass of tRNA (25,000 Da) is only about one third of the molecular mass of the aminoacyl-tRNA-EF-Tu GTP complex (70,000 Da) the complex formation should become apparent by an increase of the line widths of the imino resonances. This was indeed observed with a complex of yeast Phe-tRNA and EF-Tu GTP from T. thermophilus (Fig. 19.14). 

The spectra recorded immediately after complex formation, display resonance signals which are broadened by a factor of about 2.6 in comparison to free tRNA. In addition, it should be kept in mind that during the acquisition of the spectrum (about 1 hour), part of the GTP has already been cleaved to GDP [75]. In the GDP bound form, however, the affinity of EF-Tu for aminoacyl-tRNA is considerably lower than in the GTP bound form (Tab. 19.2 c). Table 19.2c Variation of EF-Tu using Tyr-tRNA (AEDANS).  

The imino spectrum of the Phe-tRNA -EF-Tu GTP complex also reveals several distinct line shifts in comparison to the spectrum of the free tRNA. Moreover, some of the imino resonances, namely the ones of base pairs 1 through 3 of the acceptor stem, seem to be missing. However, it cannot be excluded that these lines overlap with other lines, due to larger shift changes. The assumption of vanishing imino resonances is also supported by the work of Heerschap et al. [76], who observed a disappearance of the imino resonances of the first acceptor stem base pairs upon complex formation of tRNA with E. coli EF-Tu. 

As the ternary complex dissociates in the course of the GTP hydrolysis and the Phe-tRNA deacylates, the line width of the imino resonances decreases again and the chemical shifts adopt the positions they had before in the free tRNA. However, even after corn- 

Such an orthoester can be formed either with the oxygen atoms of both, the 2 and the 3 -hydroxyl groups of the A76 ribose (Fig. 19.16a) or with a nucleophilic functional group of the protein (Fig. 19.16 b). The structure in Fig. 19.16 a seems to be reasonable as during the (relatively slow) transacylation reaction of the amino acid residue this intermediate state has to be passed [77]. The stmcture shown in Fig. 19.16b would explain the observed chemical shift as well. In this case a nucleophile leading to an easily cleavable transient bond should be provided by the protein. A deprotonated carboxylate located in the interface between domains I and II of EF-Tii [1] is the most obvious candidate for such a function. 

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