» Articles » PMID: 33300043

Reconstitution of Mammalian Mitochondrial Translation System Capable of Correct Initiation and Long Polypeptide Synthesis from Leaderless MRNA

Overview
Specialty Biochemistry
Date 2020 Dec 10
PMID 33300043
Citations 15
Authors
Affiliations
Soon will be listed here.
Abstract

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

Citing Articles

Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics.

Bibel B, Raskar T, Couvillion M, Lee M, Kleinman J, Takeuchi-Tomita N Nucleic Acids Res. 2025; 53(3).

PMID: 39907106 PMC: 11795202. DOI: 10.1093/nar/gkaf046.


Selection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation.

Lee M, Wakigawa T, Jia Q, Liu C, Huang R, Huang S Nucleic Acids Res. 2025; 53(3).

PMID: 39878211 PMC: 11775629. DOI: 10.1093/nar/gkaf021.


Unraveling the roles and mechanisms of mitochondrial translation in normal and malignant hematopoiesis.

Liu L, Shao M, Huang Y, Qian P, Huang H J Hematol Oncol. 2024; 17(1):95.

PMID: 39396039 PMC: 11470598. DOI: 10.1186/s13045-024-01615-9.


Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics.

Bibel B, Raskar T, Couvillion M, Lee M, Kleinman J, Takeuchi-Tomita N bioRxiv. 2024; .

PMID: 39229136 PMC: 11370408. DOI: 10.1101/2024.08.21.609012.


The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches.

Brischigliaro M, Kruger A, Moran J, Antonicka H, Ahn A, Shoubridge E Nucleic Acids Res. 2024; 52(16):9710-9726.

PMID: 39036954 PMC: 11381339. DOI: 10.1093/nar/gkae645.


References
1.
Pietromonaco S, Denslow N, OBrien T . Proteins of mammalian mitochondrial ribosomes. Biochimie. 1991; 73(6):827-35. DOI: 10.1016/0300-9084(91)90062-6. View

2.
Suzuki T, Terasaki M, Hanada T, Ueda T, Wada A, Watanabe K . Proteomic analysis of the mammalian mitochondrial ribosome. Identification of protein components in the 28 S small subunit. J Biol Chem. 2001; 276(35):33181-95. DOI: 10.1074/jbc.M103236200. View

3.
Johanson U, AEvarsson A, Liljas A, Hughes D . The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants. J Mol Biol. 1996; 258(3):420-32. DOI: 10.1006/jmbi.1996.0259. View

4.
Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K . Cell-free translation reconstituted with purified components. Nat Biotechnol. 2001; 19(8):751-5. DOI: 10.1038/90802. View

5.
Richter-Dennerlein R, Oeljeklaus S, Lorenzi I, Ronsor C, Bareth B, Schendzielorz A . Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein. Cell. 2016; 167(2):471-483.e10. PMC: 5055049. DOI: 10.1016/j.cell.2016.09.003. View