Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Abstract

Human mitochondrial translational initiation factor 3 (IF3 mt ) has been identified from the human expressed sequence tag data base. Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones. IF3 mt is 278 amino acid residues in length. MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length. The cDNA for the predicted mature form of IF3 mt was cloned, and the protein was expressed in Escherichia coli in a His-tagged form. The mature form of IF3 mt has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3 mt homologous to prokaryotic factors ranges between 21-26% identical to the bacterial proteins. Purified IF3 mt promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2 mt ), [35 S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3 mt shifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability of E. coli initiation factor 1 to stimulate initiation complex formation on E. coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2 mt and IF3 mt .Mammalian mitochondria synthesize 13 polypeptides that are essential for oxidative phosphorylation. These 13 proteins are translated from nine monocistronic and two dicistronic mRNAs with overlapping reading frames (1, 2). The proteinsynthesizing system of mammalian mitochondria has a number of interesting features not observed in prokaryotes or the cell cytoplasm (3). The mRNAs in this organelle have an almost complete lack of 5Ј-and 3Ј-untranslated nucleotides. The start codon is generally located within three nucleotides of the 5Ј end of the mRNA (1, 4). Thus, mammalian mitochondrial ribosomes do not recognize the start codon using the Shine/Dalgarno interaction between the mRNA and the 16 S rRNA as observed in prokaryotes. Further, this system does not use a cap-binding and scanning mechanism such as observed in the eukaryotic cytoplasm.Three translational initiation factors, IF1, IF2, and IF3, 1 are required for the initiation of protein synthesis in bacteria (5-7). Prior to the present report, the homolog of only one of these factors, IF2 mt , had been identified, cloned, and characterized in mammalian mitochondria (8 -12). Similar to its prokaryotic counterpart, IF2 mt promotes the binding of fMet-tRNA to the small subunit of mitochondrial ribosomes in response to synthetic polynucleotides such as poly(A,U,G).The current report describes the identification and initial characterization of the mammalian mitochondrial factor equivalent to IF3. In prokaryotes IF3 has a number of roles in the initiation of protein synthesis. IF3 b...

Lack of secondary structure characterizes the 5′ ends of mammalian mitochondrial mRNAs

“…IF3 functions to dissociate intact ribosomes into their constituent subunits, in both the prokaryotic and mitochondrial systems. In prokaryotes, IF3 antagonizes initiation of leaderless mRNAs, which require intact 70S ribosomes (Udagawa et al 2004), whereas IF3 mt stimulates initiation complex formation in the presence of mitochondrial 55S ribosomes (Koc and Spremulli 2002). This stimulation is consistent with the ability of IF3 mt to dissociate 55S ribosomes and, thereby, to increase the concentration of initiation-active 28S subunits (Koc and Spremulli 2002).…”

Translation Initiation inSaccharomyces cerevisiaeMitochondria: Functional Interactions Among Mitochondrial Ribosomal Protein Rsm28p, Initiation Factor 2, Methionyl-tRNA-Formyltransferase and Novel Protein Rmd9p

“…Divergent homologs of bacterial IF3 are present in mammals and in the fission yeast Schizosaccharomyces pombe (Bhargava and Spremulli 2005). No clear homologs are present in budding yeasts, although a possible candidate of questionable significance has been reported to exist in the S. cerevisiae genome (Koc and Spremulli 2002). In contrast, genes encoding proteins homologous to Rsm28p can be identified in genomes of species closely related to S. cerevisiae, such as Kluyveromyces lactis, Candida glabrata, and Ashbya gossypii, but appear to be absent in S. pombe.…”

“…Analysis of homologous genes in the genomes of other fungal species is largely consistent with this possibility. No genes encoding potential mitochondrial proteins homologous to bacterial IF1 have been identified in eukaryotes (Koc and Spremulli 2002), although genes coding divergent forms of this short protein could go undetected. Divergent homologs of bacterial IF3 are present in mammals and in the fission yeast Schizosaccharomyces pombe (Bhargava and Spremulli 2005).…”

The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria

“…The ligated plasmids were transformed into Stratagene's E. coli DH5α cells. Candidate plasmids were confirmed by sequencing and transformed into E. coli BL21 (DE3) RIL cells (Stratagene) for expression (Koc et al, 2002).…”