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Updated: Dec 18, 2025
Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
Published on: May 10, 2018
Distinct pre-initiation steps in human mitochondrial translation
Anas Khawaja1,2, Yuzuru Itoh1,3, Cristina Remes4
1Department of Medical Biochemistry and Biophysics, Division of Molecular Metabolism, Karolinska Institutet, Biomedicum, 171 65, Solna, Sweden.
Abstract:
Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.
Insights
Human mitochondria use unique initiation factors (mtIF2, mtIF3) for protein synthesis. New research details two key assembly steps involving mS37, revealing how these factors form pre-initiation complexes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Mitochondrial translation initiation in humans involves specialized factors (mtIF2, mtIF3) distinct from bacterial systems.
- Understanding these unique factors is crucial for comprehending mitochondrial protein synthesis.
Purpose of the Study:
- To elucidate the distinct steps in mitochondrial pre-initiation complex assembly.
- To characterize the roles of mtIF2, mtIF3, and mS37 in initiating mitochondrial translation.
Main Methods:
- Single-particle cryo-electron microscopy (cryo-EM) to visualize complex structures.
- Fluorescence cross-correlation spectroscopy (FCCS) to analyze complex assembly dynamics.
Main Results:
- Identified two distinct mitochondrial pre-initiation assembly steps.
- Mitochondria-specific protein mS37 and mtIF3 stabilize the small mitoribosomal subunit for mtIF2 binding.
- mtIF3 promotes assembly independently of mRNA and initiator tRNA, with its domains mediating exclusion.
- Large mitoribosomal subunit association is essential for recruiting initiator tRNA and leaderless mRNA.
Conclusions:
- Revealed novel mechanisms of mitochondrial translation initiation in mammals.
- Detailed the sequential assembly pathway of the mitochondrial initiation complex.
- Highlighted the unique roles of mtIF3 and mS37 in regulating mitoribosome function.
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