DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic translation reinitiation is a complex process involving ribosome scanning and translation of downstream ORFs.
• Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are known to promote translation reinitiation.

Purpose of the Study:

• To elucidate the structural basis of DENR-MCTS1 interaction and its role in translation reinitiation.
• To identify key residues in DENR and MCTS1 essential for complex formation and function.

Main Methods:

• X-ray crystallography to determine the structure of MCTS1 bound to a DENR fragment.
• Site-directed mutagenesis to investigate the functional importance of specific amino acid residues.
• In vivo assays in human cells to assess the impact on translation reinitiation.

Main Results:

• The crystal structure revealed MCTS1 bound to a DENR fragment, highlighting specific interaction interfaces.
• DENR residues Glu42, Tyr43, and Tyr46 were identified as critical for MCTS1 binding.
• MCTS1 residue Phe104 was found to be essential for tRNA binding.
• Mutational analysis confirmed that both DENR-MCTS1 dimerization and tRNA binding are necessary for promoting translation reinitiation.

Conclusions:

• Specific amino acid residues in DENR and MCTS1 directly link to their molecular functions in the complex.
• The DENR-MCTS1 complex can bind tRNA independently of the ribosome, suggesting a role in recruiting tRNA during reinitiation.
• This mechanism may be analogous to the function of the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.