Assembly and functionality of the ribosome with tethered subunits

Nature Communications
Nikolay A AleksashinAlexander S Mankin

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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Citations

Sep 4, 2019·Nature Communications·Erik D CarlsonMichael C Jewett
Jul 10, 2019·Life·Pol Arranz-GibertFarren J Isaacs
Nov 11, 2019·Nature Communications·Joongoo LeeMichael C Jewett
Nov 30, 2019·Nucleic Acids Research·Michael J HammerlingMichael C Jewett
Feb 6, 2020·Nucleic Acids Research·Anne E d'AquinoMichael C Jewett
Apr 22, 2020·Nature Communications·Nikolay A AleksashinAlexander S Mankin
May 6, 2021·Nucleic Acids Research·Celine Fabret, Olivier Namy
Oct 15, 2019·Biochemistry·Fred R WardJamie H D Cate
Sep 26, 2021·Nature Communications·Fan LiuAhmed H Badran

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Datasets Mentioned

BETA
MG1655
U00096.3

Methods Mentioned

BETA
PCR
RNA-seq
Ribo-seq
GTPase
electrophoresis

Software Mentioned

Kallisto
statsmodels
Ribo
MaxQuant
HISAT2
Sleuth
Cutadapt
oRibo

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