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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach
12:12

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Published on: March 12, 2017

Quantitative characterization of Tob interactions provides the thermodynamic basis for translation

Lin Ruan1, Masanori Osawa, Nao Hosoda

  • 1Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.

The Journal of Biological Chemistry
|July 3, 2010
PubMed

Insights

The Tob-Caf1 complex regulates mRNA decay by competing with translation termination factors for binding to poly(A)-binding protein 1. This competition, influenced by protein levels, dictates deadenylation activity in eukaryotic cells.

Area of Science:

  • Molecular Biology
  • RNA Biology
  • Biochemistry

Background:

  • Translation termination-coupled deadenylation is a critical step in eukaryotic mRNA decay.
  • The deadenylases Ccr4-Caf1 and Pan2 are key players in this process.
  • Caf1 associates with Tob, which recruits it to the poly(A) tail via cytoplasmic poly(A)-binding protein 1 (PABPC1).

Purpose of the Study:

  • To elucidate the molecular mechanism regulating deadenylation.
  • To investigate the binding affinities and cellular levels of Tob, Caf1, and eRF3.
  • To understand the competition between Tob and eRF3 for PABPC1 binding.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to characterize protein interactions.
  • Western blot analysis to determine protein cellular levels.
  • Biochemical assays to measure binding affinities (K(d)).

Main Results:

  • Human Tob protein has a structured N-terminal BTG-Tob domain binding Caf1 and an unstructured C-terminal region with two PAM2 motifs binding PABPC1 (K(d) = 20 microM).
  • Cellular levels of eRF3 (4-5 microM) are significantly higher than Tob (<0.2 microM) in HeLa cells.
  • These findings support a competitive binding model for Tob and eRF3 to PABPC1.

Conclusions:

  • A thermodynamic mechanism for translation termination-coupled deadenylation mediated by the Tob-Caf1 complex is proposed.
  • The differential cellular concentrations of Tob and eRF3 likely play a crucial role in regulating deadenylation.
  • This study provides molecular insights into the interplay between translation termination and mRNA decay pathways.

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