Ribosome selectivity and nascent chain context in modulating the incorporation of fluorescent non-canonical amino acid into proteins

被引:3
|
作者
Thommen, Michael [1 ]
Draycheva, Albena [1 ]
Rodnina, Marina, V [1 ]
机构
[1] Max Planck Inst Multidisciplinary Sci, Dept Phys Biochem, Gottingen, Germany
基金
欧洲研究理事会;
关键词
SITE-SPECIFIC INCORPORATION; PEPTIDE-BOND FORMATION; ELONGATION-FACTOR TU; CELL-FREE TRANSLATION; TRANSFER-RNA; ESCHERICHIA-COLI; GENETIC-CODE; DELIVERY; BINDING; FRET;
D O I
10.1038/s41598-022-16932-7
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNA(Cys) into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNA(Cys) can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNA(Cys) in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions -1 to -4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.
引用
收藏
页数:14
相关论文
共 50 条
  • [21] Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells
    Bartoschek, Michael D.
    Ugur, Enes
    Nguyen, Tuan-Anh
    Rodschinka, Geraldine
    Wierer, Michael
    Lang, Kathrin
    Bultmann, Sebastian
    NUCLEIC ACIDS RESEARCH, 2021, 49 (11) : E62
  • [22] Non-Canonical Amino Acid Incorporation into AAV5 Capsid Enhances Lung Transduction in Mice
    Chang, Hao
    Du, Ailing
    Ren, Lingzhi
    Liu, Nan
    Zhou, Xuntao
    Gao, Guangping
    Wang, Dan
    MOLECULAR THERAPY, 2023, 31 (04) : 552 - 553
  • [23] Synthesis and incorporation of 1,2-alkanolamine-functionalized lysine as a non-canonical amino acid into GFP
    Rowlett, Chesley
    Morse, Jared
    Liu, Wenshe
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 2018, 255
  • [24] Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice
    Chang, Hao
    Du, Ailing
    Jiang, Jun
    Ren, Lingzhi
    Liu, Nan
    Zhou, Xuntao
    Liang, Jialing
    Gao, Guangping
    Wang, Dan
    MOLECULAR THERAPY METHODS & CLINICAL DEVELOPMENT, 2023, 31
  • [25] Defining TNF-α and IL-1β induced nascent proteins: Combining bio-orthogonal non-canonical amino acid tagging and proteomics
    Choi, Ka-Yee
    Lippert, Dustin N. D.
    Ezzatti, Peyman
    Mookherjee, Neeloffer
    JOURNAL OF IMMUNOLOGICAL METHODS, 2012, 382 (1-2) : 189 - 195
  • [26] Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides
    Cui, Zhenling
    Johnston, Wayne A.
    Alexandrov, Kirill
    FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, 2020, 8
  • [27] Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
    Nickling, Jessica H.
    Baumann, Tobias
    Schmitt, Franz-Josef
    Bartholomae, Maike
    Kuipers, Oscar P.
    Friedrich, Thomas
    Budisa, Nediljko
    JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, 2018, (135):
  • [28] Incorporation of Non-Canonical Amino Acids into Antimicrobial Peptides: Advances, Challenges, and Perspectives
    Du, Yuhui
    Li, Li
    Zheng, Yue
    Liu, Jiaheng
    Gong, Julia
    Qiu, Zekai
    Li, Yanni
    Qiao, Jianjun
    Huo, Yi-Xin
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2022, 88 (23)
  • [29] Probing the active site of alanine racemase by incorporation of non-canonical amino acids
    Jacobs, Kim J.
    Wang, Yane-Shih
    Liu, Wenshe
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 2011, 241
  • [30] Development of Multivalent Conjugates with a Single Non-Canonical Amino Acid
    Burrow, Naya K.
    Gourdie, Robert K.
    King, Elizabeth A.
    Travis, Christopher R.
    Goff, Cameron M.
    Nimmo, Zachary M.
    Berg, Justin M.
    Boyt, Emily L.
    Young, Douglas D.
    CHEMBIOCHEM, 2023, 24 (23)