TY - JOUR
T1 - Assembly and functionality of the ribosome with tethered subunits
AU - Aleksashin, Nikolay A.
AU - Leppik, Margus
AU - Hockenberry, Adam J.
AU - Klepacki, Dorota
AU - Vázquez-Laslop, Nora
AU - Jewett, Michael C.
AU - Remme, Jaanus
AU - Mankin, Alexander S.
N1 - Funding Information:
The authors thank Amira Kefi for help with the initial bioinformatics analysis, Teresa Szal for assistance with some of the experiments, and Cedric Orelle for advice with some experimental procedures. The authors are grateful to Maxim Svetlov and Yury Polikanov for the critical reading of the manuscript. This work was supported by the NIH grant R35 GM127134 (to A.S.M.), Institutional Research Funding projects of the Estonian Ministry of Education and Research [IUT20-21] (to J.R.), the National Science Foundation (NSF) grant MCB-1716766 (to M.C.J.), the Human Frontiers Science Program RGP0015/2017 (to M.C.J.), the David and Lucile Packard Foundation (to M.C.J.), the Camille Dreyfus Teacher-Scholar Program (to M.C.J.).
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
AB - Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
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U2 - 10.1038/s41467-019-08892-w
DO - 10.1038/s41467-019-08892-w
M3 - Article
C2 - 30804338
AN - SCOPUS:85062103842
SN - 2041-1723
VL - 10
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 930
ER -