Translational Activity of Elongation Factor Tu

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We examined the translational activity of EF-Tu by using the PURE system, in vitro translation system derived from E.coli that was contains all the components essential for translation (17). Purified of wild-type EF-Tu from Synechocystis that had been treated with various concentrations of H2O2 were added to the translation system, which constructed without EF-Tu, and observed the synthesis of DHFR. The results revealed that the synthesis of DHFR was significantly decreased upon the increasing of H2O2 concentration (Fig. 1A). Only 0.1 mM H2O2 treated EF-Tu can inactivate the translational activity by decreased approximately 80% when compared with reduced EF-Tu (Fig. 1B). The results indicated that the translational activity of EF-Tu is hyper-sensitive to H2O2. By contrast, when we monitored translation in vitro of the mutated EF-Tu, which has the replacement of Cys82 by serine (isosteric to cysteine), on the sensitivity to oxidation by H2O2 showed the synthesis of DHFR was unaffected in the presence of H2O2 (Fig. 1C). Even though we added the concentration of H2O2 to 2 mM into the Cys82 mutant protein, the translational activity was quite constant, whereas the translational activity of wild-type EF-Tu was inactivation (Fig. 1B and 1D). These results suggested that the substitution of Cys82 by serine rendered EF-Tu insensitive to inactivation by H2O2 in translational machinery.

We monitored the redox state of Cys82 of EF-Tu by thiol-modification assay that modifying free thiol group with maleimidyl reagent (average molecular mass, 5 kDa). Modification of thiol group of reduced EF-Tu with this reagent resulted in the change of electrophoretic mobility of EF-Tu on a non-reducing SDS-PAGE by the molecular mass was increase when co...

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...wn as the G domain. It is involved in the formation of several hydrogen bonds with water molecules that coordinates the Mg2+ ion in the nucleotide binding pocket (22). Moreover, Cys81 is presence in the vicinity of the site of EF-Tu interaction with the 3′ end and 5′ end of the aminoacyl-tRNA molecule (23,24). The supporting data previously revealed that chemical modification of Cys82 by N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) in Thermus thermophilus EF-Tu (25,26), equivalent to Cys 81 of EF-Tu from E.coli (27,28) and Bacillus stearothermophilus (29), interfere with the formation of an appropriate pocket on EF-Tu to accommodate the aa-tRNA for ternary complex. Substitution of Cys82 by alanine prevents alkylation of EF-Tu by TosPheCH2Cl and allowed the interaction with aa-tRNA, suggesting that Cys82 plays a more specific role in the former conformation.

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