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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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Ribosomes01:27

Ribosomes

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Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that synthesize large quantities of protein—such as secretory cells in the human pancreas—can contain millions of ribosomes.
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Ribosomes01:27

Ribosomes

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Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that synthesize large quantities of protein—such as secretory cells in the human pancreas—can contain millions of ribosomes.
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Ribosomal RNA Synthesis02:53

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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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Improving Translational Accuracy02:07

Improving Translational Accuracy

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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing
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In vitro ribosome synthesis and evolution through ribosome display.

Michael J Hammerling1, Brian R Fritz1, Danielle J Yoesep1

  • 1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208, USA.

Nature Communications
|March 1, 2020
PubMed
Summary
This summary is machine-generated.

Directed evolution of ribosomes is now possible in vitro using the novel Ribosome Synthesis and Evolution (RISE) method. This technique overcomes cell viability limitations, enabling the selection of functional ribosomal RNA (rRNA) variants and mutant ribosomes with new properties.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Synthetic Biology

Background:

  • Directed evolution of ribosomes is hindered by the need for cell viability, limiting mutation possibilities.
  • Developing novel ribosome functions requires overcoming these cellular constraints.

Purpose of the Study:

  • To develop a cell-free, in vitro methodology for ribosome synthesis and evolution (RISE).
  • To enable directed evolution of ribosomes independent of cell viability.
  • To facilitate the selection of ribosomes with expanded substrate incorporation and novel functions.

Main Methods:

  • Combined cell-free synthesis and assembly of translationally competent ribosomes.
  • Utilized ribosome display for a fully in vitro evolution system.
  • Screened large libraries of ribosomal RNA (rRNA) variants.

Main Results:

  • Successfully selected active rRNA genotypes from a library of ~1.7 × 10^7 variants.
  • Identified clindamycin-resistant mutant ribosomes from a library of ~4 × 10^3 variants.
  • Demonstrated the prevalence of positive epistasis in resistant genotypes, crucial for new function selection.

Conclusions:

  • The Ribosome Synthesis and Evolution (RISE) method provides a powerful platform for in vitro ribosome evolution.
  • RISE overcomes cell viability limitations, enabling broader exploration of ribosome function.
  • This methodology will advance the understanding of molecular translation and facilitate the engineering of novel ribosome properties.