Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP-and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H 2 O 2 , whereas GDPbound EF-Tu was resistant to H 2 O 2 . The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H 2 O 2 , confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Oxidation of Translation Factor EF-Tu Inhibits the Repair of Photosystem II

“…Oxidized EF-G and EF-Tu can be reduced and reactivated by thioredoxin, a small redox protein that regulates the activity of target proteins by reducing disulfide bonds. This observation suggests that reducing power from photosynthetic electron transport might be transmitted to EF-G and EF-Tu via thioredoxin in vivo (Kojima et al, 2009;Nishiyama et al, 2011;Yutthanasirikul et al, 2016). Interactions of thioredoxin with EF-G and EF-Tu in Synechocystis were also suggested by results of studies using thioredoxin-affinity chromatography (Lindahl and Florencio, 2003) and similar results were obtained with spinach (Spinacia olerace) chloroplasts (Balmer et al, 2003).…”

“…PCC 6803 (hereafter Synechocystis) showed that ROS inhibit the elongation step in the translation of psbA mRNA, which encodes the D1 protein (Nishiyama et al, 2001(Nishiyama et al, , 2004. Biochemical studies revealed that two translation factors, EF-G and EF-Tu, which are responsible for translational elongation, are the targets of ROS within the translational machinery of Synechocystis (Kojima et al, 2007;Yutthanasirikul et al, 2016). EF-G translocates peptidyl-tRNA from the A site to the P site of the ribosome, and it is inactivated by H 2 O 2 via oxidation of Cys-105 and Cys-242, with subsequent formation of an intramolecular disulfide bond (Kojima et al, 2009).…”

“…EF-G translocates peptidyl-tRNA from the A site to the P site of the ribosome, and it is inactivated by H 2 O 2 via oxidation of Cys-105 and Cys-242, with subsequent formation of an intramolecular disulfide bond (Kojima et al, 2009). EF-Tu delivers aminoacyl-tRNA to the A site of the ribosome, and it is inactivated by H 2 O 2 via oxidation of a single Cys residue, Cys-82, with subsequent formation of both sulfenic acid and an intermolecular disulfide bond (Yutthanasirikul et al, 2016). Oxidized EF-G and EF-Tu can be reduced and reactivated by thioredoxin, a small redox protein that regulates the activity of target proteins by reducing disulfide bonds.…”

Formation of oxidised (OX) proteins inEntamoeba histolyticaexposed to auranofin and consequences on the parasite virulence

“…During the elongation stage of translation, the GTP dependent eEF1A delivers aminoacyl‐tRNA to the A‐site of the 80S ribosome. Elongation factors are prone to oxidation due to the presence of cysteine residues that are essential for their translational activity (Liu et al, ; Nagano et al, ; Yutthanasirikul et al, ). According to the results of protein synthesis in AF‐treated parasites, we can exclude that oxidation of eEF1A in E. histolytica inhibits protein synthesis.…”

Oxygen and ROS in Photosynthesis

“…ROS have a strong inhibitory effect on translation in cyanobacteria [348]. The mechanism of the inhibition by H 2 O 2 is the oxidation of cysteine residues and the subsequent formation of an intramolecular disulfide bond in translation elongation factor G [348], and the formation of a sulfenic acid and an intermolecular disulfide bond in elongation factor Tu [349,350]. The inhibition of translational elongation exerts its effect on the activity of PSII by inhibiting or slowing down the turnover of the D1 protein [351].…”