Assembly and functionality of the ribosome with tethered subunits

Nikolay A. Aleksashin; Margus Leppik; Adam J. Hockenberry; Dorota Klepacki; Nora Vázquez-Laslop; Michael C. Jewett; Jaanus Remme; Alexander S. Mankin

doi:10.1038/s41467-019-08892-w

Assembly and functionality of the ribosome with tethered subunits

Nikolay A. Aleksashin, Margus Leppik, Adam J. Hockenberry, Dorota Klepacki, Nora Vázquez-Laslop, Michael C. Jewett, Jaanus Remme, Alexander S. Mankin^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

33 Scopus citations

Abstract

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.

Original language	English (US)
Article number	930
Journal	Nature communications
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/s41467-019-08892-w
State	Published - Dec 1 2019

Funding

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.).

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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abstract = "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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AU - Vázquez-Laslop, Nora

AU - Jewett, Michael C.

AU - Remme, Jaanus

AU - Mankin, Alexander S.

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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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Assembly and functionality of the ribosome with tethered subunits

Abstract

Funding

ASJC Scopus subject areas

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