SELECTIVE PHOTO-OXIDATION OF HISTIDINE-RESIDUES IN POLYPEPTIDE-CHAIN ELONGATION-FACTOR TU FROM ESCHERICHIA-COLI

When E. coli elongation factor Tu [EF-Tu] was photooxidized for 20 min at 0.degree. C in the presence of 10 .mu.M GDP and 5 .mu.M rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. These activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA.cntdot.EF-Tu.cntdot.GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu.cntdot.GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. No histidine residues were lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu.cntdot.GDP was partially degraded with trypsin; each of the resulting tryptic fragments (D, B and C) was analyzed for histidine content. Fragments B, C and D lost 0.7, 0.5 and 0.2 residues of histidine, respectively. Fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, suggesting that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.