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Interaction of Escherichia coli EF-Tu.GTP and EF-Tu.GDP with analogues of the 3' terminus of aminoacyl-tRNA.

The affinity of chemically synthetized analogues of the 3′ terminus of phenylalanyl-tRNA and leucyl-tRNA for the aminoacyl-tRNA binding site of Escherichia coli elongation factor-Tu · GTP complex (EF-Tu · GTP) increases in the order A(Phe)°C-A(Leu)<pA(Phe)≤C-A(Phe)<amino-acyl-tRNA [2′(3′)-O-l-phenylalanyladenosine<cytidylyl-(3′≤5′)-2′(3′)-O-l-leucyladenosine≤2′(3′)-0-l-phenylalanyladenosine 5′-phosphate<cytidylyl-(3′ 5′)-2′(3′)-0-l-phenylalanyladenosine<aminoacyl-tRNA], as we revealed using the l-l-tosylamido-2-phenylethyl chloromethyl ketone assay [(1979) FEES Lett. 93, 329-332]. By contrast, C-A(d-Phe), C-A(fPhe), and an equimolar mixture of C-A+phenylalanyl methyl ester are at least twenty times less active than C-A(Phe) and do not differ from C-A alone; puromycin is almost inactive. The affinity of A(Phe), pA(Phe), C-A(Phe) and C-A(Leu) (just like the affinity of the intact molecule of aminoacyl-tRNA) for EF-Tu · GDP is substantially lower than for EF-Tu · GTP. However, with puromycin, C-A, C-A(o-Phe), C-A(fPhe) and an equimolar mixture of C-A+phenylalanvl methyl ester this difference between EF-Tu · GTP and EF-Tu · GDP is no longer observed or is almost insignificant.



These results indicate that the binding site for the 3′terminus of aminoacyi-tRNA in EF-Tu · GTP is intimately involved in the fundamental property of the factor to discriminate between aminoacyl-tRNA, peptidyl-tRNA and tRNA. The presence of the undisturbed 2′(3′)-O-l-aminoacyladenosine structure at the 3′ end of respective ligands appears to be a prerequisite for the recognition of the ligand by the aminoacyl-tRNA binding site of EF-Tu · GTP. In addition, this site apparently also contains regions which interact with the phosphate residue and with cytidine of the 3′-terminal C-A sequence of aminoacyl-tRNA and can contribute significantly to the stability of the interaction. The results support the idea of the key role of the 3′-terminal C-C-A structure of tRNA as ‘general ligand’ in protein synthesis.



Finally, our results indicate that the occupation of the guanosine nucleotide binding site of EF-Tu either by GTP or GDP could control the affinity ofthe factor for the whole molecule of aminoacyl-tRNA simply by modulating the affinity of EF-Tu just for the 3′ terminus of the poly-nucleotide.