*Corresponding author: Yoshitaka Nishiyama,

Transcription

1 SUPPLEMENTAL DATA Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli Takanori Nagano 1, Kouji Kojima 1,7, Toru Hisabori 2, Hidenori Hayashi 3, Eugene Hayato Morita 4, Takashi Kanamori 5,8, Tomoko Miyagi 5,8, Takuya Ueda 5,8, and Yoshitaka Nishiyama 1,6, * From 1 Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama , Japan; 2 Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama , Japan; 3 Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama , Japan; 4 Department of Bioresources, Faculty of Agriculture, Ehime University, Tarumi, Matsuyama, Ehime , Japan; 5 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Chiba , Japan; 6 Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama , Japan *Corresponding author: Yoshitaka Nishiyama, nishiyama@molbiol.saitama-u.ac.jp Present address: 7 Department of Biological Chemistry, Chubu University, 1200 Kasugai , Japan. 8 GeneFrontier Corporation, 308 Todai-Kashiwa Venture Plaza, Kashiwanoha, Kashiwa, Chiba , Japan. EXPERIMENTAL PROCEDURES Circular Dichroism Spectrometry The ultraviolet-circular dichroism (UV-CD) spectra of reduced and oxidized EF-G were obtained with a UV-CD spectrophotometer (J-820; JASCO, Tokyo, Japan), equipped with a temperature control unit (PTC-423L; JASCO). EF-G at 1.6 µm was incubated at 25ºC for 5 min in the presence of 5 mm DTT or 0.5 mm H 2 O 2 prior to the measurement. Measurements were performed at 4ºC with 8 scans. The width of band and the spectral resolution were 1 nm, and the scan speed was 100 nm min -1. LEGENDS TO FIGURES FIGURE S1. Effects of various oxidants on the redox state and the translational activity of wild-type EF-G. Wild-type EF-G (2 µm) was incubated for 10 min in the presence, separately, of 0.5 mm H 2 O 2, 10 µm CuCl 2, and 10 µm Aldrithiol TM -4. EF-G was then subjected to the 1

2 thiol-modification assay (A) and the translational assay (B), as described in the legend to Figure 1. No, No thiol modification; Red, reducing conditions. FIGURE S2. Effects of H 2 O 2 on the secondary structure of wild-type EF-G. The UV-CD spectra of wild-type EF-G (1.6 µm) that had been incubated for 5 min in the presence of 5 mm DTT (blue line) and 0.5 mm H 2 O 2 (red line). FIGURE S3. Mass spectra of tryptic digests of wild-type EF-G. The acquired profiles of wild-type EF-G, which had been incubated with 2 mm H 2 O 2 (A) and 5 mm DTT (B) were focused on mass ranges from to Incubated proteins were digested with trypsin and the resultant peptides were analyzed by MALDI-TOF MS. C, D, and E, Magnified views of the peaks of peptide fragments that contained Cys114, Cys266, and Cys398, respectively. F, Magnified view of the spectrum of the peptide fragments that resulted from the combination of two peptides via formation of a disulfide bond between Cys114 and Cys266. FIGURE S4. Predicted three-dimensional structure of EF-G of E. coli. The crystal structure of EF-G of E. coli, which was bound to the ribosome-recycling factor (RRF), was determined by Yokoyama et al. (19). The structure of EF-G was generated by subtracting the structural information of RRF from that of the EF-G-RRF complex (PDB: 3JOE). The putative positions of three cysteine residues in EF-G are shown in yellow. FIGURE S5. Alignment of the amino acid sequences and locations of cysteine residues in bacterial, chloroplastic and mitochondrial EF-G proteins. Primary amino acid sequences of EF-G were analyzed with the Clustal X software. Amino acid sequences of EF-G from cyanobacteria and Arabidopsis thaliana were obtained from Cyanobase ( and TAIR ( respectively. Other sequences were obtained from UniProtKB/Swiss-Prot ( The organisms examined were as follows: Escherichia coli (ECOLI_P0A6M8, fusa); Synechocystis sp. PCC 6803 (S68_Slr1463); Synechococcus sp. PCC 7942 (S79_Syc0655d); Thermosynechococcus elongatus BP-1 (Tel_Tlr1749); Anabaena sp. PCC 7120 (A71_All4338); Synechococcus sp. WH 8102 (S81_SYNW2137); Gloeobacter violaceus PCC 7421 (GLOVI_Q7NEF2); Prochlorococcus marinus MIT 9313 (PROMM_Q7V501); Salmonella typhi CT18 (SALTY_P0A1H4); Rhodobacter sphaeroides strain ATCC (RHOSP_A4WVL1); Bradyrhizobium japonicum (BRAJA_Q89J81); Bacillus subtilis (BACSU_P80868); Thermus thermophilus (THETH_Q72I01); Arabidopsis thaliana (ARATH_AT1G62750); Glycine max (SOYBN_P34811); Oryza sativa (ORYZA_Q7XQQ7); Homo sapiens (HUMAN_Q8N6D8); Mus musculus (Mouse_Q8K0D5) and Drosophila melanogaster (DROME_Q9VM33). Domains of EF-G are indicated by boxes with names 2

3 at the top. Dark vertical bands in boxes indicate locations of cysteine residues in EF-G. The positions of cysteine residues in EF-G (ECOLI_P0A6M8) are numbered. 3

4

5

6

7

8