Citation: Peng Sijia, Sun Ruirui, Wang Wenjuan, Chen Chunlai. Single-molecule FRET studies on interactions between elongation factor 4 (LepA) and ribosomes[J]. Chinese Chemical Letters, ;2018, 29(10): 1503-1508. doi: 10.1016/j.cclet.2017.12.006 shu

Single-molecule FRET studies on interactions between elongation factor 4 (LepA) and ribosomes

a.
School of Life Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing 100084, China
b.
School of Life Sciences and Technology Center for Protein Sciences, Tsinghua University, Beijing 100084, China

Corresponding author: Wang Wenjuan, wangwenjuan@biomed.tsinghua.edu.cn Chen Chunlai, chunlai@mail.tsinghua.edu.cn
Received Date: 2 December 2017
Revised Date: 20 December 2017
Accepted Date: 21 December 2017
Available Online: 8 October 2017

Figures(5)

Elongation factor 4 (EF4) is one of the highly conserved translational GTPases, whose functions are largely unknown. Structures of EF4 bound ribosomal PRE-translocation and POST-translocation complexes have both been visualized. On top of cellular, structural, and biochemical studies, several controversial models have been raised to rationalize functions of EF4. However, how EF4 modulates elongation through its interactions with ribosomes has not been revealed. Here, using single-molecule fluorescence resonance energy transfer assays, we directly captured short-lived EF4·GTP bound ribosomal PRE and POST translocation complexes, which may adopt slightly different conformations from structures prepared using GDP, GDPNP, or GDPCP. Furthermore, we revealed that EF4·GTP severely impairs delivery of aminoacyl-tRNA into the A-site of the ribosome and moderately accelerates translocation. We proposed that functions of EF4 are to slow overall elongation and to stall majority of ribosomes in POSTstates under stress conditions.
- Ribosome,
- Single-molecule biophysics,
- Translation,
- Fluorescence resonance energy transfer,
- GTPase
1. [1]
  T.M. Schmeing, V. Ramakrishnan, Nature 461(2009) 1234-1242. doi: 10.1038/nature08403
2. [2]
  T. Margus, M. Remm, T. Tenson, BMC Genom. 8(2007) 15. doi: 10.1186/1471-2164-8-15
3. [3]
  R. Ero, V. Kumar, Y. Chen, Y.G. Gao, RNA Biol. 13(2016) 1258-1273. doi: 10.1080/15476286.2016.1201627
4. [4]
  Y. Qin, N. Polacek, O. Vesper, et al., Cell 127(2006) 721-733. doi: 10.1016/j.cell.2006.09.037
5. [5]
  N.J. Dibb, P.B. Wolfe, J. Bacteriol. 166(1986) 83-87. doi: 10.1128/jb.166.1.83-87.1986
6. [6]
  M. Pech, Z. Karim, H. Yamamoto, et al., Proc. Natl. Acad. Sci. U. S. A. 108(2011) 3199-3203. doi: 10.1073/pnas.1012994108
7. [7]
  F. Yang, Y. Gao, Z. Li, et al., Biochim. Biophys. Acta 1837(2014) 1674-1683. doi: 10.1016/j.bbabio.2014.05.353
8. [8]
  F. Yang, Z. Li, J. Hao, Y. Qin, Protein Cell 5(2014) 563-567. doi: 10.1007/s13238-014-0050-3
9. [9]
  S. Shoji, B.D. Janssen, C.S. Hayes, K. Fredrick, Biochimie 92(2010) 157-163. doi: 10.1016/j.biochi.2009.11.002
10. [10]
  L. Li, Y. Hong, G. Luan, et al., mBio 5(2014) e01708.
11. [11]
  R.N. Evans, G. Blaha, S. Bailey, T.A. Steitz, Proc. Natl. Acad. Sci. U. S. A.105(2008) 4673-4678. doi: 10.1073/pnas.0801308105
12. [12]
  S.R. Connell, M. Topf, Y. Qin, et al., Nat. Struct. Mol. Biol. 15(2008) 910-915. doi: 10.1038/nsmb.1469
13. [13]
  D. Zhang, K. Yan, G. Liu, et al., Nat. Struct. Mol. Biol. 23(2016) 125-131. doi: 10.1038/nsmb.3160
14. [14]
  M.G. Gagnon, J. Lin, T.A. Steitz, Proc. Natl. Acad. Sci. U. S. A. 113(2016) 4994-4999. doi: 10.1073/pnas.1522932113
15. [15]
  M.G. Gagnon, J. Lin, D. Bulkley, T.A. Steitz, Science 345(2014) 684-687. doi: 10.1126/science.1253525
16. [16]
  V. Kumar, R. Ero, T. Ahmed, et al., J. Biol. Chem. 291(2016) 12943-12950. doi: 10.1074/jbc.M116.725945
17. [17]
  P.E. March, M. Inouye, J. Biol. Chem. 260(1985) 7206-7213.
18. [18]
  P.E. March, M. Inouye, Proc. Natl. Acad. Sci. U. S. A. 82(1985) 7500-7504. doi: 10.1073/pnas.82.22.7500
19. [19]
  H.Q. Liu, C.L. Chen, H.B. Zhang, et al., Proc. Natl. Acad. Sci. U. S. A. 108(2011) 16223-16228. doi: 10.1073/pnas.1103820108
20. [20]
  R. Balakrishnan, K. Oman, S. Shoji, R. Bundschuh, K. Fredrick, Nucleic Acids Res. 42(2014) 13370-13383. doi: 10.1093/nar/gku1098
21. [21]
  M.R. Gibbs, K.M. Moon, M. Chen, et al., Proc. Natl. Acad. Sci. U. S. A. 114(2017) 980-985. doi: 10.1073/pnas.1613665114
22. [22]
  H.Q. Liu, D.L. Pan, M. Pech, B.S. Cooperman, J. Mol. Biol. 396(2010) 1043-1052. doi: 10.1016/j.jmb.2009.12.043
23. [23]
  S. Shoji, S.E. Walker, K. Fredrick, Mol. Cell. 24(2006) 931-942. doi: 10.1016/j.molcel.2006.11.025
24. [24]
  A.L. Konevega, N. Fischer, Y.P. Semenkov, et al., Nat. Struct. Mol. Biol.14(2007) 318-324. doi: 10.1038/nsmb1221
25. [25]
  S.K. Chakkarapani, G. Park, S.H. Kang, Chin. Chem. Lett. 26(2015) 1490-1495. doi: 10.1016/j.cclet.2015.10.017
26. [26]
  A.M. van Oijen, N.E. Dixon, Nat. Struct. Mol. Biol. 22(2015) 948-952. doi: 10.1038/nsmb.3119
27. [27]
  C.J. Bustamante, C.M. Kaiser, R.A. Maillard, D.H. Goldman, C.A. Wilson, Annu. Rev. Biophys. 43(2014) 119-140. doi: 10.1146/annurev-biophys-051013-022811
28. [28]
  C.E. Aitken, A. Petrov, J.D. Puglisi, Annu. Rev. Biophys. 39(2010) 491-513. doi: 10.1146/annurev.biophys.093008.131427
29. [29]
  G. Park, S.K. Chakkarapani, S. Ju, S. Ahn, S.H. Kang, Chin. Chem. Lett.29(2018) 505-508. doi: 10.1016/j.cclet.2017.08.018
30. [30]
  F.F. Li, J.N. Gu, X.C. Zhou, Chin. Chem. Lett. 26(2015) 1514-1517. doi: 10.1016/j.cclet.2015.09.013
31. [31]
  H. Cai, C. Zhou, Q. Yang, et al., Chin. Chem. Lett. 29(2018) 531-534. doi: 10.1016/j.cclet.2017.09.010
32. [32]
  C.L. Chen, B. Stevens, J. Kaur, et al., Mol. Cell. 42(2011) 367-377. doi: 10.1016/j.molcel.2011.03.024
33. [33]
  C.L. Chen, H.B. Zhang, S.L. Broitman, et al., Nat. Struct. Mol. Biol. 20(2013) 582-588. doi: 10.1038/nsmb.2544
34. [34]
  C.W. Lin, A.Y. Ting, J. Am. Chem. Soc. 128(2006) 4542-4543. doi: 10.1021/ja0604111
35. [35]
  J. Yin, A.J. Lin, D.E. Golan, C.T. Walsh, Nat. Protoc. 1(2006) 280-285. doi: 10.1038/nprot.2006.43
36. [36]
  S. Leng, Q.L. Qiao, Y. Gao, et al., Chin. Chem. Lett. 28(2017) 1911-1915. doi: 10.1016/j.cclet.2017.03.034
37. [37]
  L. Peng, Y. Xu, P. Zou, Chin. Chem. Lett. 28(2017) 1925-1928. doi: 10.1016/j.cclet.2017.09.037
38. [38]
  F. Sun, W.-B. Zhang, Chin. Chem. Lett. 28(2017) 2078-2084. doi: 10.1016/j.cclet.2017.08.052
39. [39]
  E.I. De Laurentiis, H.J. Wieden, Sci. Rep. 5(2015) 8573. doi: 10.1038/srep08573
40. [40]
  J. Chen, A. Petrov, A. Tsai, S.E. O'Leary, J.D. Puglisi, Nat. Struct. Mol. Biol. 20(2013) 718-727. doi: 10.1038/nsmb.2567
41. [41]
  C. Chen, X. Cui, J.F. Beausang, et al., Proc. Natl.Acad. Sci. U. S. A.113(2016) 7515-7520. doi: 10.1073/pnas.1602668113
42. [42]
  T. Pape, W. Wintermeyer, M.V. Rodnina, Embo. J. 17(1998) 7490-7497. doi: 10.1093/emboj/17.24.7490
43. [43]
  W. Liu, C. Chen, D. Kavaliauskas, et al., Nucleic Acids Res. 43(2015) 9519-9528. doi: 10.1093/nar/gkv856
44. [44]
  M.V. Rodnina, A. Savelsbergh, V.I. Katunin, W. Wintermeyer, Nature 385(1997) 37-41. doi: 10.1038/385037a0
45. [45]
  A. Korostelev, D.N. Ermolenko, H.F. Noller, Curr. Opin. Chem. Biol. 12(2008) 674-683. doi: 10.1016/j.cbpa.2008.08.037
46. [46]
  J.B. Munro, K.Y. Sanbonmatsu, C.M.T. Spahn, S.C. Blanchard, Trends Biochem. Sci. 34(2009) 390-400. doi: 10.1016/j.tibs.2009.04.004
47. [47]
  J. Frank, R.L. Gonzalez, Annu. Rev. Biochem. 79(2010) 381-412. doi: 10.1146/annurev-biochem-060408-173330
48. [48]
  N. Fischer, A.L. Konevega, W. Wintermeyer, M.V. Rodnina, H. Stark, Nature 466(2010) 329-333. doi: 10.1038/nature09206
49. [49]
  X. Agirrezabala, H.Y. Liao, E. Schreiner, et al., Proc. Natl. Acad. Sci. U. S. A. 109(2012) 6094-6099. doi: 10.1073/pnas.1201288109
50. [50]
  D.S. Tourigny, I.S. Fernandez, A.C. Kelley, V. Ramakrishnan, Science 340(2013) 1235490. doi: 10.1126/science.1235490
51. [51]
  J. Zhou, L. Lancaster, J.P. Donohue, H.F. Noller, Science 340(2013) 1236086. doi: 10.1126/science.1236086
52. [52]
  A. Pulk, J.H.D. Cate, Science 340(2013) 1235970. doi: 10.1126/science.1235970
53. [53]
  A.F. Brilot, A.A. Korostelev, D.N. Ermolenko, N. Grigorieff, Proc. Natl. Acad. Sci. U. S. A. 110(2013) 20994-20999. doi: 10.1073/pnas.1311423110
54. [54]
  J. Zhou, L. Lancaster, J.P. Donohue, H.F. Noller, Science 345(2014) 1188-1191. doi: 10.1126/science.1255030
55. [55]
  E. Salsi, E. Farah, J. Dann, D.N. Ermolenko, Proc.Natl.Acad. Sci. U. S. A.111(2014) 15060-15065. doi: 10.1073/pnas.1410873111
56. [56]
  E. Salsi, E. Farah, Z. Netter, J. Dann, D.N. Ermolenko, J. Mol. Biol. 427(2015) 454-467. doi: 10.1016/j.jmb.2014.11.010
57. [57]
  C.L. Chen, B. Stevens, J. Kaur, et al., Proc. Natl. Acad. Sci. U. S. A. 108(2011) 16980-16985. doi: 10.1073/pnas.1106999108
58. [58]
  T.M. Schmeing, R.M. Voorhees, A.C. Kelley, et al., Science 326(2009) 688-694. doi: 10.1126/science.1179700
59. [59]
  J.L.E. Heller, R. Kamalampeta, H.J. Wieden, Mol. Cell Biol. 37(2017) e00653-16.
60. [60]
  A.L. Starosta, J. Lassak, K. Jung, D.N. Wilson, FEMS Microbiol. Rev. 38(2014) 1172-1201. doi: 10.1111/1574-6976.12083
61. [61]
  E. Giudice, R. Gillet, Trends Biochem. Sci. 38(2013) 403-411. doi: 10.1016/j.tibs.2013.06.002
62. [62]
  H. Himeno, N. Nameki, D. Kurita, A. Muto, T. Abo, Biochimie 114(2015) 102-112. doi: 10.1016/j.biochi.2014.11.014
1. [1]
  Siwei Wang , Wei-Lei Zhou , Yong Chen . Cucurbituril and cyclodextrin co-confinement-based multilevel assembly for single-molecule phosphorescence resonance energy transfer behavior. Chinese Chemical Letters, 2024, 35(12): 110261-. doi: 10.1016/j.cclet.2024.110261
2. [2]
  Shuai Qiu , Jia He , Xiao Hu , Hongxia Yan , Zhao Gao , Wei Tian . Cation-π enhanced triplet-to-singlet Förster resonance energy transfer for fluorescence afterglow. Chinese Chemical Letters, 2025, 36(4): 110057-. doi: 10.1016/j.cclet.2024.110057
3. [3]
  Jun-Jie Fang , Yun-Peng Xie , Xing Lu . Organooxotin and cobalt/manganese heterometallic nanoclusters exhibiting single-molecule magnetism. Chinese Journal of Structural Chemistry, 2025, 44(4): 100515-100515. doi: 10.1016/j.cjsc.2025.100515
4. [4]
  Xinyi Luo , Ke Wang , Yingying Xue , Xiaobao Cao , Jianhua Zhou , Jiasi Wang . Digital PCR-free technologies for absolute quantitation of nucleic acids at single-molecule level. Chinese Chemical Letters, 2025, 36(2): 109924-. doi: 10.1016/j.cclet.2024.109924
5. [5]
  Yinling HOU , Jia JI , Hong YU , Xiaoyun BIAN , Xiaofen GUAN , Jing QIU , Shuyi REN , Ming FANG . A rhombic Dy₄-based complex showing remarkable single-molecule magnet behavior. Chinese Journal of Inorganic Chemistry, 2025, 41(3): 605-612. doi: 10.11862/CJIC.20240251
6. [6]
  Xiaoyao Ma , Jinling Zhang , Ge Fang , He Gao , Jie Gao , Li Fu , Yuanyuan Hou , Gang Bai . Förster resonance energy transfer reveals phillygenin and swertiamarin concurrently target AKT on different binding domains to increase the anti-inflammatory effect. Chinese Chemical Letters, 2024, 35(5): 108823-. doi: 10.1016/j.cclet.2023.108823
7. [7]
  Zhaorui Song , Qiulian Hao , Bing Li , Yuwei Yuan , Shanshan Zhang , Yongkuan Suo , Hai-Hao Han , Zhen Cheng . NIR-Ⅱ fluorescence lateral flow immunosensor based on efficient energy transfer probe for point-of-care testing of tumor biomarkers. Chinese Chemical Letters, 2025, 36(1): 109834-. doi: 10.1016/j.cclet.2024.109834
8. [8]
  Er-Meng Wang , Ziyi Wang , Xu Ban , Xiaowei Zhao , Yanli Yin , Zhiyong Jiang . Chemoselective photocatalytic sulfenylamination of alkenes with sulfenamides via energy transfer. Chinese Chemical Letters, 2024, 35(12): 109843-. doi: 10.1016/j.cclet.2024.109843
9. [9]
  Qiuye Wang , Yabing Sun , Liangxue Lai , Haijing Cui , Yonglong Ye , Ming Yang , Weihao Zhu , Bo Yuan , Quanliang Mao , Wenzhi Ren , Aiguo Wu . MMP-9-responsive probe for fluorescence-magnetic resonance dual-mode imaging of hepatocellular carcinoma models with different metastatic capacities. Chinese Chemical Letters, 2025, 36(4): 110212-. doi: 10.1016/j.cclet.2024.110212
10. [10]
  Zhao-Xia Lian , Xue-Zhi Wang , Chuang-Wei Zhou , Jiayu Li , Ming-De Li , Xiao-Ping Zhou , Dan Li . Producing circularly polarized luminescence by radiative energy transfer from achiral metal-organic cage to chiral organic molecules. Chinese Chemical Letters, 2024, 35(8): 109063-. doi: 10.1016/j.cclet.2023.109063
11. [11]
  Chaoqun Ma , Yuebo Wang , Ning Han , Rongzhen Zhang , Hui Liu , Xiaofeng Sun , Lingbao Xing . Carbon dot-based artificial light-harvesting systems with sequential energy transfer and white light emission for photocatalysis. Chinese Chemical Letters, 2024, 35(4): 108632-. doi: 10.1016/j.cclet.2023.108632
12. [12]
  Huanyu Liu , Gang Yu , Ruoyao Guo , Hao Qi , Jiayin Zheng , Tong Jin , Zifeng Zhao , Zuqiang Bian , Zhiwei Liu . Direct identification of energy transfer mechanism in Ce^Ⅲ-Mn^Ⅱ system by constructing molecular heteronuclear complexes. Chinese Chemical Letters, 2025, 36(2): 110296-. doi: 10.1016/j.cclet.2024.110296
13. [13]
  Weixu Li , Yuexin Wang , Lin Li , Xinyi Huang , Mengdi Liu , Bo Gui , Xianjun Lang , Cheng Wang . Promoting energy transfer pathway in porphyrin-based sp2 carbon-conjugated covalent organic frameworks for selective photocatalytic oxidation of sulfide. Chinese Journal of Structural Chemistry, 2024, 43(7): 100299-100299. doi: 10.1016/j.cjsc.2024.100299
14. [14]
  Yan Fan , Jiao Tan , Cuijuan Zou , Xuliang Hu , Xing Feng , Xin-Long Ni . Unprecedented stepwise electron transfer and photocatalysis in supramolecular assembly derived hybrid single-layer two-dimensional nanosheets in water. Chinese Chemical Letters, 2025, 36(4): 110101-. doi: 10.1016/j.cclet.2024.110101
15. [15]
  Hui Liu , Xiangyang Tang , Zhuang Cheng , Yin Hu , Yan Yan , Yangze Xu , Zihan Su , Futong Liu , Ping Lu . Constructing multifunctional deep-blue emitters with weak charge transfer excited state for high-performance non-doped blue OLEDs and single-emissive-layer hybrid white OLEDs. Chinese Chemical Letters, 2024, 35(10): 109809-. doi: 10.1016/j.cclet.2024.109809
16. [16]
  Ruike Hu , Kangmin Wang , Junxiang Liu , Jingxian Zhang , Guoliang Yang , Liqiu Wan , Bijin Li . Extended π-conjugated systems by external ligand-assisted C−H olefination of heterocycles: Facile access to single-molecular white-light-emitting and NIR fluorescence materials. Chinese Chemical Letters, 2025, 36(4): 110113-. doi: 10.1016/j.cclet.2024.110113
17. [17]
  Ali Dai , Zhiguo Zheng , Liusheng Duan , Jian Wu , Weiming Tan . Small molecule chemical scaffolds in plant growth regulators for the development of agrochemicals. Chinese Chemical Letters, 2025, 36(4): 110462-. doi: 10.1016/j.cclet.2024.110462
18. [18]
  Ruotong Wei , Aokun Liu , Jian Kuang , Zhiwen Wang , Lu Yu , Changlin Tian . Probing the dynamic properties in the LLPS process via site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy. Chinese Chemical Letters, 2025, 36(4): 110029-. doi: 10.1016/j.cclet.2024.110029
19. [19]
  Kun Zhang , Ni Dan , Dan-Dan Ren , Ruo-Yu Zhang , Xiaoyan Lu , Ya-Pan Wu , Li-Lei Zhang , Hong-Ru Fu , Dong-Sheng Li . A small D-A molecule with highly heat-resisting room temperature phosphorescence for white emission and anti-counterfeiting. Chinese Journal of Structural Chemistry, 2024, 43(3): 100244-100244. doi: 10.1016/j.cjsc.2024.100244
20. [20]
  Jin Wang , Xiaoyan Pan , Junyu Zhang , Qingqing Zhang , Yanchen Li , Weiwei Guo , Jie Zhang . Active molecule-based theranostic agents for tumor vasculature normalization and antitumor efficacy. Chinese Chemical Letters, 2024, 35(8): 109187-. doi: 10.1016/j.cclet.2023.109187

Get Citation

PDF

XML

Metrics

PDF Downloads(2)
Abstract views(785)
HTML views(11)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142