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) O‐l‐phenylalanyladenosine O‐l‐leucyladenosine≤2′(3′)‐0‐l‐phenylalanyladenosine 5′‐phosphate 0‐l‐phenylalanyladenosine 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.