Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1

Abstract

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2 mt and IF3 mt , instead of the three that are present in eubacteria. The mammalian IF2 mt possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2 mt in complex with initiator fMet-tRNA i Met and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2 mt mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.protein synthesis | ribosome-IF2mt complex | cryo-EM structure | molecular modeling P rotein synthesis entails a complex series of events in which the ribosome interacts with a number of ligands and translation factors so as to conduct an efficient, accurate, and well-regulated process (1). The first event in translation is the initiation step, which requires the formation of an initiation complex, composed of the small ribosomal subunit (30S, in eubacteria), mRNA, initiator tRNA (fMet-tRNA i Met ) and three initiation factors (IF1, IF2, and IF3) (2). IF3 helps in preparation of the 30S initiation complex by preventing premature docking of the large subunit (50S, in eubacteria) (3, 4). IF2 promotes the binding of the initiator tRNA to the peptidyl (P) site of the 30S subunit and facilitates the association of the 50S subunit to form the 70S initiation complex (5). Initiation factor IF1, together with IF2, stabilizes the 30S initiation complex (6). IF1 also plays a role in translation initiation fidelity in conjunction with IF3 (7-9). The binding site of IF1 overlaps with that of the aminoacyl-tRNA on the 30S subunit (the A site), thereby making this site unavailable for the initiator tRNA during translation initiation (10-13). However, no equivalent of eubacterial IF1 has been detected in mammalian mitochondria (14); mitochondrial protein synthesis requires only two initiation factors, IF2 mt and IF3 mt .There is a lack of consensus in the nomenclature used to describe the domains of IF2 by biochemists (15-18) and structural biologists (12,19). A uniform nomenclature that addresses both structural and functional considerations was used in our previous study (20). According to this nomenclature the approximately 98 kDa Escherichia coli IF2 is composed of six domains (Fig. 1A). Domains I and II are not present in all of the eubacterial factors, are not highly conserved, and are also absent in the mammalian IF2 mt . Domain VI is divided into two structura...

Mechanism of protein biosynthesis in mammalian mitochondria

“…To assess whether the 37 amino acid insertion in IF2 mt interacts with the same binding site on the ribosome, cryo-electron microscopy was performed on an E. coli initiation complex formed with 70S ribosomes, IF2 mt , fMet-tRNA, mRNA and GDPNP [63]. IF2 mt makes extensive contacts with the interface sides of both ribosomal subunits extending from the lower body of the ribosome to the decoding center (Fig.…”

“…Although electron density for this region of IF2 mt could be observed, no structural information on homologous proteins is known for this domain preventing the development of an accurate model for this region of the factor. The model for the remainder of IF2 mt was based on the crystal structure of Methanobacterium thermoautotrophicum aIF2 and the NMR structures of the VIC1 and VIC2 domains of Bacillus (now Geobacillus ) sterarothermophilus [63]. In this model, IF2 mt resembles the structure of the archaeal and eubacterial factors with the exception of the 37 amino acid insertion.…”

“…B. Model for the 3-D structure of IF2 mt , based on the cryo-EM map of IF2 mt [63]. Domain 3 has been omitted from the structure, since cryo-EM images were not of sufficient resolution to obtain its coordinates, and no corresponding structures were available in the databases upon which to build a model.…”

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit

“…Mitochondrial initiation factor 3 (IF3 mt ) promotes the dissociation of the mitochondrial ribosome [7] and reduces the binding of fMet-tRNA to the small subunit in the absence of mRNA [8]. No factor equivalent to IF1 has been observed in mammalian mitochondria and a short segment in IF2 mt is thought to play the role of IF1 in this system [9,10]. …”

Taurine-containing Uridine Modifications in tRNA Anticodons Are Required to Decipher Non-universal Genetic Codes in Ascidian Mitochondria

“…This single tRNA Met can be charged with Met and then formylated by mitochondrial transformylase (42,43). Then, the formylmethionyl(fMet)-tRNA Met is recognized by mitochondrial initiation factor 2 and used as an initiator tRNA (44,45). As Met-tRNA Met is not fully formylated by mitochondrial transformylase in mitochondria, Met-tRNA Met is also used as an elongator tRNA.…”