Protein Biosynthesis in Mitochondria: Past Simple, Present Perfect, Future Indefinite.

Mitochondria are obligate organelles of most eukaryotic cells that perform many different functions important for cellular homeostasis. The main role of mitochondria is supplying cells with energy in a form of ATP, which is synthesized in a chain of oxidative phosphorylation reactions on the organelle inner membrane. It is commonly believed now that mitochondria have the endosymbiotic origin. In the course of evolution, they have lost most of their genetic material as a result of genome reduction and gene transfer to the nucleus. The majority of mitochondrial proteins are synthesized in the cytosol and then imported to the mitochondria. However, almost all known mitochondria still contain genomes that are maintained and expressed. The processes of protein biosynthesis in the mitochondria - mitochondrial translation - substantially differs from the analogous processes in bacteria and the cytosol of eukaryotic cells. Mitochondrial translation is characterized by a high degree of specialization and specific regulatory mechanisms. In this review, we analyze available information on the common principles of mitochondrial translation with emphasis on the molecular mechanisms of translation initiation in the mitochondria of yeast and mammalian cells.

[40 Years of Studying RNA Import into Mitochondria: From Basic Mechanisms to Gene Therapy Strategies].

Mitochondria of many living species internalize nuclear DNA-encoded ribonucleic acids. The pools of imported RNA molecules, as well as fine mechanisms of these processes, are highly species-specific. To date, baker's yeast Saccharomyces cerevisiae are the best studied in this regard. Moreover, the processes of yeast RNA mitochondrial import have been the basis of modeling several gene therapy strategies aimed to palliate negative effects of pathogenic mutations in human mitochondrial DNA. In this review, we summarize our current knowledge about the molecular events taking place in course of yeast RNA import into mitochondria. Also, we describe how this process can be used for compensation of pathogenic mutations in mitochondrial genomes of humans.

Mitochondrial Translation Initiation Factor 3: Structure, Functions, Interactions, and Implication in Human Health and Disease.

Mitochondria are essential organelles of eukaryotic cell that provide its respiratory function by means of the electron transfer chain. Expression of mitochondrial genes is organized in a bacterial-like manner; however multiple evolutionary differences are observed between the two systems, including translation initiation machinery. This review is dedicated to the mitochondrial translation initiation factor 3 (IF3mt), which plays a key role in the protein synthesis in mitochondria. Involvement of IF3mt in human health and disease is discussed.

Interaction between Saccharomyces cerevisiae Mitochondrial DNA-Binding Protein Abf2p and Cce1p Resolvase.

Mitochondrial DNA is susceptible to the action of reactive oxygen species generated by the reactions of oxidative phosphorylation. Homologous recombination is one of the mechanisms providing integrity of the mitochondrial genome. Some proteins that take part in this process in budding yeast mitochondria have been identified. These include Abf2p, the major protein of the mt-nucleoid that specifically binds cruciform DNA, and Cce1p - Holliday junction resolvase. Here we show that Abf2p does not significantly affect either binding of Cce1p to branched DNA or rate and specificity of Holliday junction resolution. These data suggest the existence of an alternative homologous recombination pathway in yeast mitochondria.

Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.

Noncanonical functions of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases, together with their main function of covalent binding of an amino acid to a corresponding tRNA, also perform many other functions. They take part in regulation of gene transcription, apoptosis, translation, and RNA splicing. Some of them function as cytokines or catalyze different reactions in living cells. Noncanonical functions can be mediated by additional domains of these proteins. On the other hand, some of the noncanonical functions are directly associated with the active center of the aminoacylation reaction. In this review we summarize recent data on the noncanonical functions of aminoacyl-tRNA synthetases and on the mechanisms of their action.

Specific features of 5S rRNA structure - its interactions with macromolecules and possible functions.

Small non-coding RNAs are today a topic of great interest for molecular biologists because they can be regarded as relicts of a hypothetical "RNA world" which, apparently, preceded the modern stage of organic evolution on Earth. The small molecule of 5S rRNA (approximately 120 nucleotides) is a component of large ribosomal subunits of all living beings (5S rRNAs are not found only in mitoribosomes of fungi and metazoans). This molecule interacts with various protein factors and 23S (28S) rRNA. This review contains the accumulated data to date concerning 5S rRNA structure, interactions with other biological macromolecules, intracellular traffic, and functions in the cell.

[Directed import of macromolecules into mitochondria].

Mitochondria are multifunctional organelles of eukaryotic cells that provide the energy for the cells by oxidative phosphorylation, play an important role in the apoptosis and take part in Fe-S clusters formation, fatty acids oxidation and synthesis of some aminoacids. They contain their own genome and are able to transcribe and to translate it. However, the vast majority of the macromolecules which function inside the mitochondria are imported into these organelles from the cytoplasm. The imported macromolecules include proteins and several types of small RNAs. Protein import is a universal process and its mechanism is conserved among all species. This mechanism is now known in detail. RNA import was shown to occur in several groups of eukaryotes, while the pool of imported RNA molecules varies in different organisms. Although the knowledge about the mechanisms of RNA import is less extensive than for the proteins, it becomes clear that these mechanisms are not universal among all the species possessing this pathway. In this review, we summarize the data about the import of macromolecules mentioned above into mitochondria.

Cotranslational folding of globin.

Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.

Non-random usage of 'degenerate' codons is related to protein three-dimensional structure.

We report an analysis of a novel sequence-structure database of mammalian proteins incorporating nucleotide sequences of the exon regions of their genes together with protein sequence and structural information. We find that synonymous codon families (i.e. coding the same residue) have non-random codon distribution frequencies between protein secondary structure types. Their structural preferences are related to the third, 'silent' nucleotide position in a codon. We also find that some synonymous codons show very different or even opposite structural preferences at the N- or C-termini of structure fragments, relative to those observed for their amino acid residues.

Mitochondrial import of a yeast cytoplasmic tRNA (Lys): possible roles of aminoacylation and modified nucleosides in subcellular partitioning.

The yeast tRNA(CUU)LYS is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97-98%) and mitochondria (2-3%). We have optimized the conditions for its specific import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA-protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed.

[Import of cytoplasmic lysyl tRNA in mitochondria of baker's yeasts: prospects for study in vivo and in vitro]. / Import tsitoplazmaticheskoi lizinovoi tRNK v mitokhondrii pekarskikh drozhzhei: vozmozhnost' izucheniia in vivo i in vitro.

To study the mechanisms of targeting tRNA(CUULys) from the cytoplasm into the mitochondrial compartment of the yeast cells, two test systems have been developed. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm; however, only tRNA(CUULys) was found in the mitochondrial compartment. The in vitro import of this tRNA into isolated mitochondria required the presence of ATP and soluble cellular proteins in the reaction mixture. Two protein fractions were found to be necessary to direct the import in vitro. The first one had a high heparin-binding affinity, while the other one was not retained on heparin-Sepharose. Aminoacylation of the tRNA(Lys) before the transport and/or addition of the correspondent lysyl-tRNA synthetase to the in vitro system increased the efficiency of the import, but the protein fraction with heparin-binding properties is still required. The unmodified transcripts of the tRNA(CUULys) gene were shown to be able to be transported into isolated yeast mitochondria.

Cotranslational heme binding to nascent globin chains.

Globin synthesis in cell-free extracts of rabbit reticulocytes was carried out in the presence of 3H-labeled hemin. Sucrose gradient centrifugation analysis revealed [3H]hemin in the polyribosome fraction. The addition of puromycin resulted in the release of both [3H]hemin- and [14C]leucine-labeled polypeptide from the polyribosomes. The data suggest cotranslational folding of the globin molecule on the ribosome and cotranslational heme binding to the nascent globin chain.

A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis.

The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (pI 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleosomes, thus mimicking the processes occurring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes.

Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a contranslational protein-folding model.

Examination of nascent globin peptides accumulating in vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of alpha- and beta-globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.

Loosened nucleosome linker folding in transcriptionally active chromatin of chicken embryo erythrocyte nuclei.

We have investigated the mechanism of the electrophoresis-driven chromatin aggregation which had been described by Weintraub (1984, Cell 38, 17-27) as a putative mean for propagation of genetic repression in eukaryotes. We show that the oligonucleosome aggregates are assembled de novo at the starting zone of DNP electrophoresis. A new system of native two-dimensional DNP electrophoresis has been worked out to separate the oligonucleosome aggregates ('A' particles) and the freely-migrating oligonucleosomes ('B' particles). The 'B' particle fraction which is derived from transcriptionally-active chromatin regions undergoes an extensive nuclease degradation of its DNA termini during the nuclease digestion. This fraction is partially depleted of histones H1 and H5 and is enriched in HMG nonhistone proteins. 'A' particles comprise the repressed chromatin DNA fragments which are about 60 b.p. longer than the corresponding DNA oligomers of 'B' particles. An oligonucleosome preparation containing the elongated DNA oligomers has been also isolated by means of sucrose gradient ultracentrifugation. Exonuclease III mapping reveals that the two chromatin fractions differ by an extent of terminal linker DNA trimming during the Micrococcal nuclease digestion rather than by the nucleosome repeat length. The complex character of nuclease digestion is not observed when the chromatin is digested in solution after the nuclear lysis. We argue that the protection of terminal oligonucleosome linkers is due to selective condensation of inactive chromatin in chicken erythrocyte nuclei and that the terminal DNA tails together with linker histones bound to them mediate the aggregation of repressed chromatin fragments.

[Role of the code redundancy in determining cotranslational protein folding]. / Rol' vyrozhdennosti koda v opredelenii puti kotransliatsionnogo svorachivaniia belka.

It has been demonstrated earlier in our laboratory that rare codon clusters can determine the boundaries of the polypeptide chain fragments of the same secondary structure type during the co-translational protein folding. According to this data, co-translational protein folding can occur under condition of a correlation between the frequency of codon choice in mRNAs and the relative abundance of their isoaccepting tRNAs. The alterations in the spectrum and concentrations of the isoaccepting tRNAs in different cells were demonstrated by many authors. The existence of a mechanism of the coordinate regulation of the levels (activities) of the isoaccepting tRNAs, corresponding aminoacyl-tRNA synthetases and mRNAs predominantly translated at a given moment of time can be suggested. Such a mechanism can ensure the needed accuracy of the protein folding process. Analysis of gene sequences of various pro- and eukaryotic organisms carried out in the present work revealed that the codon usage frequency spectra of simultaneously synthesized proteins are similar. The relative appearance of the most rare and frequent codons in investigated gene sequences displays a high degree of conservatism. It has also been found that structural-homologous proteins from different organisms (cytochromes c, myoglobins) have very similar codon frequency distribution profiles. This property retains despite the significant variations in the codon usage spectra in the investigated gene sequences. The data obtained indicate that the codon distribution in mRNAs whose diversity is mainly conditioned by the genetic code redundance is a program that determines translational rates of different mRNA parts thus controlling the spatial folding of the synthesized peptide chain.

[Mechanism of formation of associated oligonucleosomes during electrophoresis]. / Mekhanizm obrazovaniia assotsiatov oligonukleosom pri élektroforeze.

Here we used DNP electrophoresis to study the mechanism of formation of associated oligonucleosomes (A-particles) which have been previously shown by Weintraub to contain the DNA of silent but not of transcriptionally-active genes from chicken erythrocyte nuclei. We found out that A-particles are generated in the course of electrophoresis and that their assembly is inhibited as the result of redistribution of the most mobile fraction of histones H1 and H5. The mechanism and the conditions for the assembly of A-particles at the start line of DNP electrophoresis are discussed in the paper. The DNA molecules constituting A-particles appeared to be about 60 base pairs longer than the DNA of free oligonucleosomes. Thus in course of nuclease treatment of erythrocyte nuclei two chromatin fractions can be observed, one of them containing the DNA of transcriptionally active genes loses its terminal DNA regions owing to rapid degradation of cleaved nucleosome linkers, while the other containing the DNA of repressed genes maintains its terminal linker DNA and gives rise to the associated oligonucleosomes.