Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

Native tertiary structure and nucleoside modifications suppress tRNA's intrinsic ability to activate the innate immune sensor PKR.

Interferon inducible protein kinase PKR is an essential component of innate immunity. It is activated by long stretches of dsRNA and provides the first line of host defense against pathogens by inhibiting translation initiation in the infected cell. Many cellular and viral transcripts contain nucleoside modifications and/or tertiary structure that could affect PKR activation. We have previously demonstrated that a 5'-end triphosphate-a signature of certain viral and bacterial transcripts-confers the ability of relatively unstructured model RNA transcripts to activate PKR to inhibit translation, and that this activation is abrogated by certain modifications present in cellular RNAs. In order to understand the biological implications of native RNA tertiary structure and nucleoside modifications on PKR activation, we study here the heavily modified cellular tRNAs and the unmodified or the lightly modified mitochondrial tRNAs (mt-tRNA). We find that both a T7 transcript of yeast tRNA(Phe) and natively extracted total bovine liver mt-tRNA activate PKR in vitro, whereas native E. coli, bovine liver, yeast, and wheat tRNA(Phe) do not, nor do a variety of base- or sugar-modified T7 transcripts. These results are further supported by activation of PKR by a natively folded T7 transcript of tRNA(Phe)in vivo supporting the importance of tRNA modification in suppressing PKR activation in cells. We also examine PKR activation by a T7 transcript of the A14G pathogenic mutant of mt-tRNA(Leu), which is known to dimerize, and find that the misfolded dimeric form activates PKR in vitro while the monomeric form does not. Overall, the in vitro and in vivo findings herein indicate that tRNAs have an intrinsic ability to activate PKR and that nucleoside modifications and native RNA tertiary folding may function, at least in part, to suppress such activation, thus serving to distinguish self and non-self tRNA in innate immunity.

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

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(iMet) 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.

Analysis of the functional consequences of lethal mutations in mitochondrial translational elongation factors.

Mammalian mitochondria synthesize a set of thirteen proteins that are essential for energy generation via oxidative phosphorylation. The genes for all of the factors required for synthesis of the mitochondrially encoded proteins are located in the nuclear genome. A number of disease-causing mutations have been identified in these genes. In this manuscript, we have elucidated the mechanisms of translational failure for two disease states characterized by lethal mutations in mitochondrial elongation factor Ts (EF-Ts(mt)) and elongation factor Tu (EF-Tu(mt)). EF-Tu(mt) delivers the aminoacyl-tRNA (aa-tRNA) to the ribosome during the elongation phase of protein synthesis. EF-Ts(mt) regenerates EF-Tu(mt):GTP from EF-Tu(mt):GDP. A mutation of EF-Ts(mt) (R325W) leads to a two-fold reduction in its ability to stimulate the activity of EF-Tu(mt) in poly(U)-directed polypeptide chain elongation. This loss of activity is caused by a significant reduction in the ability of EF-Ts(mt) R325W to bind EF-Tu(mt), leading to a defect in nucleotide exchange. A mutation of Arg336 to Gln in EF-Tu(mt) causes infantile encephalopathy caused by defects in mitochondrial translation. EF-Tu(mt) R336Q is as active as the wild-type protein in polymerization using Escherichia coli 70S ribosomes and E. coli [(14)C]Phe-tRNA but is inactive in polymerization with mitochondrial [(14)C]Phe-tRNA and mitochondrial 55S ribosomes. The R336Q mutation causes a two-fold decrease in ternary complex formation with E. coli aa-tRNA but completely inactivates EF-Tu(mt) for binding to mitochondrial aa-tRNA. Clearly the R336Q mutation in EF-Tu(mt) has a far more drastic effect on its interaction with mitochondrial aa-tRNAs than bacterial aa-tRNAs.

Proteomics and electron microscopic characterization of the unusual mitochondrial ribosome-related 45S complex in Leishmania tarentolae.

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.

Characterization of the human mitochondrial methionyl-tRNA synthetase.

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu.

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).

Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for its component proteins.

The mitochondrial ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the mitochondrial ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the mitochondrial ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian mitochondrial 55S ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the ribosome. Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.

Tomato EF-Ts(mt), a functional mitochondrial translation elongation factor from higher plants.

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Ts(mt) that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Ts(mt)-GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Ts(mt) to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Ts(mt) co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Ts(mt) fraction. The purified LeEF-Ts(mt) stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Ts(mt) was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Ts(mt) encodes a functional mitochondrial EF-Ts. LeEF-Ts(mt) represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants.

RNA-binding proteins of mammalian mitochondria.

A UV-cross-linking assay was used to identify RNA-binding proteins in mammalian mitochondria. A number of these proteins were detected ranging in molecular mass from 15 to 120 kDa. All of the mRNA-binding activities were localized to the matrix except for two proteins which are primarily associated with the inner membrane. None of the polypeptides is specific for binding mitochondrial mRNAs since all bound mRNAs from other sources with comparable efficiency. Some preference for binding mRNA over tRNA or homoribopolymers was observed with several of the proteins. A protein with characteristic pentatricopeptide repeat motifs found in many RNA binding proteins was identified associated with the small subunit of the mitochondrial ribosome.