Mutational characterization and mapping of the 70S ribosome active site

d’Aquino, Anne E.; Azim, Tasfia; Aleksashin, Nikolay A.; Hockenberry, Adam J.; Krüger, Antje; Jewett, Michael C.

doi:10.1093/nar/gkaa001

Cited by 33 publications

(49 citation statements)

References 85 publications

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“…iSAT enables one-pot co-activation of rRNA transcription, assembly of rRNA with native ribosomal proteins into E. coli ribosomes, and synthesis of functional proteins from these ribosomes in a crude S150 extract lacking native ribosomes (Figure 1a, bottom). A key feature of this system is the ability to generate ribosomal variants by simply changing the DNA input, which enables rapid screening of E. coli ribosome mutations 31,32 . Moreover, because ribosomes assembled in iSAT have lower activity than in vivo-assembled versions 8,23,24 and there are known inefficiencies with ribosome reconstitution in vitro 8,23 , we hypothesized that iSAT would enable us to identify ribosomes where stabilized rRNAs could lead to improved activity.…”

Section: Eterna Participants Can Design Mutant Ribosomes With Diverse Sequences That Outperform Computationally Predicted Designsmentioning

confidence: 99%

Community science designed ribosomes with beneficial phenotypes

Krüger

Watkins

Wellington-Oguri

et al. 2021

Preprint

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Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

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Section: Eterna Participants Can Design Mutant Ribosomes With Diverse Sequences That Outperform Computationally Predicted Designsmentioning

confidence: 99%

Community science designed ribosomes with beneficial phenotypes

Krüger

Watkins

Wellington-Oguri

et al. 2021

Preprint

Self Cite

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“…Preparing the PTC mutant library. The PTC mutant library was generated by transferring individual mutations from the pT7rrnB library 44 into the 23S rRNA gene in the poRbs plasmid.…”

Section: Engineering Ribo-t-expressing Cells Sq171 Fg Cells (Supplemmentioning

confidence: 99%

A fully orthogonal system for protein synthesis in bacterial cells

et al. 2020

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Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

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“…Although testing for every possible nucleotide mutation is a daunting task, focusing on key functional regions allows one to reasonably mitigate it. For example, a first standardized and complete mutational survey was recently produced for the Peptidyl Transferase Center (PTC) [ 37 ], a region located at the core of the ribosome and associated with peptide bond formation. Totaling 180 point mutations, this study indicates that despite the highly-conserved nature of the PTC, almost every nucleotide possesses certain mutational flexibility, so one or more mutations at these positions still permit full-length protein synthesis in vitro.…”

Section: The Multiple Sources Of Heterogeneity In Ribosome Structumentioning

confidence: 99%

Structural Heterogeneities of the Ribosome: New Frontiers and Opportunities for Cryo-EM

Poitevin

Kushner

et al. 2020

Molecules

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The extent of ribosomal heterogeneity has caught increasing interest over the past few years, as recent studies have highlighted the presence of structural variations of the ribosome. More precisely, the heterogeneity of the ribosome covers multiple scales, including the dynamical aspects of ribosomal motion at the single particle level, specialization at the cellular and subcellular scale, or evolutionary differences across species. Upon solving the ribosome atomic structure at medium to high resolution, cryogenic electron microscopy (cryo-EM) has enabled investigating all these forms of heterogeneity. In this review, we present some recent advances in quantifying ribosome heterogeneity, with a focus on the conformational and evolutionary variations of the ribosome and their functional implications. These efforts highlight the need for new computational methods and comparative tools, to comprehensively model the continuous conformational transition pathways of the ribosome, as well as its evolution. While developing these methods presents some important challenges, it also provides an opportunity to extend our interpretation and usage of cryo-EM data, which would more generally benefit the study of molecular dynamics and evolution of proteins and other complexes.

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Mutational characterization and mapping of the 70S ribosome active site

Cited by 33 publications

References 85 publications

Community science designed ribosomes with beneficial phenotypes

Community science designed ribosomes with beneficial phenotypes

A fully orthogonal system for protein synthesis in bacterial cells

Structural Heterogeneities of the Ribosome: New Frontiers and Opportunities for Cryo-EM

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