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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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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.
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The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
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Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis
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Ribosome structure and dynamics by smFRET microscopy.

Bassem Shebl1, Zenia Norman1, Peter V Cornish1

  • 1Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.

Methods in Enzymology
|November 30, 2014
PubMed
Summary

Single-molecule biophysics reveals ribosome function. Single-molecule Förster resonance energy transfer (smFRET) studies detail ribosome mechanisms as a ribozyme and riboswitch.

Keywords:
RibosomeRibosome dynamicsSingle moleculeTranslationsmFRETsmFRET acquisition

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Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • The ribosome, a complex machine of RNA and protein, synthesizes all cellular protein.
  • Its peptidyl transferase center and binding sites are primarily RNA, classifying it as a ribozyme.
  • Ribosomes undergo conformational changes, functioning as riboswitches.

Purpose of the Study:

  • To explore the application of single-molecule Förster resonance energy transfer (smFRET) in studying ribosome function.
  • To detail the experimental design, from sample preparation to data analysis, for smFRET studies on ribosomes.

Main Methods:

  • Single-molecule Förster resonance energy transfer (smFRET) is utilized.
  • Detailed protocols for sample preparation, data acquisition, and analysis are described.

Main Results:

  • smFRET experiments provide insights into ribosome dynamics.
  • The study outlines the methodology for applying smFRET to investigate ribosome mechanisms.

Conclusions:

  • Single-molecule biophysics, particularly smFRET, significantly enhances understanding of ribosome function.
  • The described methods are broadly applicable to other biological systems.