Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Takanori Nagano,Kouji Kojima,Toru Hisabori,Hidenori Hayashi,Eugene Hayato Morita,Takashi Kanamori,Tomoko Miyagi,Takuya Ueda,Yoshitaka Nishiyama

doi:10.1074/jbc.m112.378067

Abstract

Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mM H(2)O(2) resulted in the complete loss of translational activity. The inactivation of EF-G by H(2)O(2) was attributable to the oxidation of two specific cysteine residues, namely, Cys(114) and Cys(266), and subsequent formation of an intramolecular disulfide bond. Replacement of Cys(114) by serine rendered EF-G insensitive to oxidation and inactivation by H(2)O(2). Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

Highlights

Elongation factor G of Escherichia coli is sensitive to oxidation
We examined the effects of reactive oxygen species (ROS) on the redox state of cysteine residues and the activity of Elongation factor G (EF-G) of E. coli, using a reconstituted translation system derived from E. coli
Effects of Oxidants on the Translational Activity of EF-G and the Redox State of Its Cysteine Residues—We examined the effects of oxidants on the translational activity of EF-G using the PURE system, a translation system in vitro derived from E. coli that was generated by mixing the individual components required for translation [12]

Summary

Background

Elongation factor G of Escherichia coli is sensitive to oxidation. Results: Elongation factor G is inactivated via the formation of an intramolecular disulfide bond. Oxidative Damage to Elongation Factor G in Translation teine residues with the resultant formation of an intramolecular disulfide bond [10, 11]. Alignment of the deduced amino acid sequences of EF-G proteins from various organisms revealed that the two cysteine residues that correspond to the targets of oxidation by ROS in Synechocystis are strongly conserved in the EF-G proteins of several species of cyanobacteria, as well as in those of nonphotosynthetic prokaryotes, such as E. coli [11]. Treatment of EF-G with H2O2 resulted in the oxidation of two specific cysteine residues and subsequent formation of an intramolecular disulfide bond This oxidation was responsible for the inactivation of EF-G in translation. Our observations suggest that regulation of translation via the redox state of EF-G might occur in photosynthetic prokaryotes and in other prokaryotes, such as E. coli

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION