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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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 helps...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...

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Related Experiment Video

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Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution
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Methodology to probe subunit interactions in ribonucleotide reductases.

A Quamrul Hassan1, Yongting Wang, Lars Plate

  • 1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Biochemistry
|November 18, 2008
PubMed
Summary

This study investigates interactions between Escherichia coli Ribonucleotide Reductase (RNR) subunits alpha2 and beta2 using modified cysteine residues. Findings reveal key interaction sites and a potential high-throughput screen for RNR inhibitors.

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Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis
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Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Ribonucleotide reductases (RNRs) are crucial enzymes for DNA synthesis and repair.
  • The Escherichia coli RNR enzyme comprises alpha2 and beta2 subunits, with interactions primarily at the beta2 C-terminus.
  • Understanding these subunit interactions is key to enzyme function and regulation.

Purpose of the Study:

  • To map the interaction sites between the alpha2 and beta2 subunits of Escherichia coli RNR.
  • To develop a method for screening potential inhibitors of RNR subunit interactions.

Main Methods:

  • Site-directed mutagenesis introduced cysteine residues into the beta2 subunit C-terminal tail.
  • Cysteine residues were modified with benzophenone (photo-cross-linker) and dimethylaminonaphthalene (fluorophore).
  • Cross-linking and fluorescence assays, SDS-PAGE, and mass spectrometry were used to analyze subunit interactions.

Main Results:

  • Benzophenone modification revealed specific sites of alpha2-beta2 cross-linking.
  • Dimethylaminonaphthalene modification showed fluorescence changes correlating with interaction strength.
  • Dissociation constants (K(d) ≈ 0.4 μM) were determined for key interaction sites.
  • Decreased fluorescence upon complex disruption suggests a high-throughput screening approach.

Conclusions:

  • The C-terminal tail of the beta2 subunit is critical for alpha2-beta2 interaction in E. coli RNR.
  • Fluorescence changes linked to subunit interactions can be used to screen for RNR inhibitors.
  • This research provides insights into RNR enzyme mechanisms and potential therapeutic targets.