Probing of nucleic acid compaction using low-frequency Raman spectroscopy.

Compaction of nucleic acids, namely DNA and RNA, determines their functions and involvement in vital cell processes including transcription, replication, DNA repair and translation. However, experimental probing of the compaction of nucleic acids is not straightforward. In this study, we suggest an approach for this probing using low-frequency Raman spectroscopy. Specifically, we show theoretically, computationally and experimentally the quantifiable correlation between the low-frequency Raman intensity from nucleic acids, magnitude of thermal fluctuations of atomic positions, and the compaction state of biomolecules. Noteworthily, we highlight that the LF Raman intensity differs by an order of magnitude for different samples of DNA, and even for the same sample in the course of long-term storage. The feasibility of the approach is further shown by assessment of the DNA compaction in the nuclei of plant cells. We anticipate that the suggested approach will enlighten compaction of nucleic acids and their dynamics during the key processes of the cell life cycle and under various factors, facilitating advancement of molecular biology and medicine.

Pentatricopeptide Protein PTCD2 Regulates COIII Translation in Mitochondria of the HeLa Cell Line.

Protein biosynthesis in mitochondria is tightly coupled with assembly of inner membrane complexes and therefore must be coordinated with cytosolic translation of the mRNAs corresponding to the subunits which are encoded in the nucleus. Molecular mechanisms underlying the regulation of mitochondrial translation remain unclear despite recent advances in structural biology. Until now, only one translational regulator of protein biosynthesis in mammalian mitochondria is known-protein TACO1, which regulates translation of COI mRNA. Here we describe the function of pentatricopeptide-containing protein PTCD2 as a translational regulator of another mitochondrially encoded subunit of cytochrome c oxidase-COIII in the HeLa cell line. Deletion of the PTCD2 gene leads to significant decrease in COIII translation efficiency and impairment in CIV activity. Additionally, we show that PTCD2 protein is partially co-sedimentates with associated mitochondrial ribosome and associates with mitochondrial ribosome proteins in pull-down assays. These data allow concluding that PTCD2 is a specific translational regulator of COIII which attracts the mRNA to the mitochondrial ribosome.

Yeast Translational Activator Mss51p and Human ZMYND17 - Two Proteins with a Common Origin, but Different Functions.

Despite its similarity to protein biosynthesis in bacteria, translation in the mitochondria of modern eukaryotes has several unique features, such as the necessity for coordination of translation of mitochondrial mRNAs encoding proteins of the electron transport chain complexes with translation of other protein components of these complexes in the cytosol. In the mitochondria of baker's yeast Saccharomyces cerevisiae, this coordination is carried out by a system of translational activators that predominantly interact with the 5'-untranslated regions of mitochondrial mRNAs. No such system has been found in human mitochondria, except a single identified translational activator, TACO1. Here, we studied the role of the ZMYND17 gene, an ortholog of the yeast gene for the translational activator Mss51p, on the mitochondrial translation in human cells. Deletion of the ZMYND17 gene did not affect translation in the mitochondria, but led to the decrease in the cytochrome c oxidase activity and increase in the amount of free F1 subunit of ATP synthase. We also investigated the evolutionary history of Mss51p and ZMYND17 and suggested a possible mechanism for the divergence of functions of these orthologous proteins.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker's yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Yeast Mitochondrial Translation Initiation Factor 3 Interacts with Pet111p to Promote COX2 mRNA Translation.

Mitochondrial genomes code for several core components of respiratory chain complexes. Thus, mitochondrial translation is of great importance for the organelle as well as for the whole cell. In yeast, mitochondrial translation initiation factor 3, Aim23p, is not essential for the organellar protein synthesis; however, its absence leads to a significant quantitative imbalance of the mitochondrial translation products. This fact points to a possible specific action of Aim23p on the biosynthesis of some mitochondrial protein species. In this work, we examined such peculiar effects of Aim23p in relation to yeast mitochondrial COX2 mRNA translation. We show that Aim23p is indispensable to this process. According to our data, this is mediated by Aimp23p interaction with the known specific factor of the COX2 mRNA translation, Pet111p. If there is no Aim23p in the yeast cells, an increased amount of Pet111p ensures proper COX2 mRNA translation. Our results demonstrate the additional non-canonical function of initiation factor 3 in yeast mitochondrial translation.

Initiation Factor 3 is Dispensable For Mitochondrial Translation in Cultured Human Cells.

The initiation of protein synthesis in bacteria is ruled by three canonical factors: IF1, IF2, and IF3. This system persists in human mitochondria; however, it functions in a rather different way due to specialization and adaptation to the organellar micro-environment. We focused on human mitochondrial IF3, which was earlier studied in vitro, but no knock-out cellular models have been published up to date. In this work, we generated human HeLa cell lines deficient in the MTIF3 gene and analyzed their mitochondrial function. Despite the lack of IF3mt in these cells, they preserved functional mitochondria capable of oxygen consumption and protein synthesis; however, the translation of ATP6 mRNA was selectively decreased which compromised the assembly of ATP synthase. Together with the analogous results obtained earlier for baker's yeast mitochondrial IF3, our findings point to a functional divergence of mitochondrial initiation factors from their bacterial ancestors.

S. cerevisiae Strain Lacking Mitochondrial IF3 Shows Increased Levels of Tma19p during Adaptation to Respiratory Growth.

After billions of years of evolution, mitochondrion retains its own genome, which gets expressed in mitochondrial matrix. Mitochondrial translation machinery rather differs from modern bacterial and eukaryotic cytosolic systems. Any disturbance in mitochondrial translation drastically impairs mitochondrial function. In budding yeast Saccharomyces cerevisiae, deletion of the gene coding for mitochondrial translation initiation factor 3 - AIM23, leads to an imbalance in mitochondrial protein synthesis and significantly delays growth after shifting from fermentable to non-fermentable carbon sources. Molecular mechanism underlying this adaptation to respiratory growth was unknown. Here, we demonstrate that slow adaptation from glycolysis to respiration in the absence of Aim23p is accompanied by a gradual increase of cytochrome c oxidase activity and by increased levels of Tma19p protein, which protects mitochondria from oxidative stress.

Biological and Evolutionary Significance of Terminal Extensions of Mitochondrial Translation Initiation Factor 3.

Protein biosynthesis in mitochondria is organized in a bacterial manner. However, during evolution, mitochondrial translation mechanisms underwent many organelle-specific changes. In particular, almost all mitochondrial translation factors, being orthologous to bacterial proteins, are characterized by some unique elements of primary or secondary structure. In the case of the organellar initiation factor 3 (IF3), these elements are several dozen amino acids long N- and C-terminal extensions. This study focused on the terminal extensions of baker's yeast mitochondrial IF3, Aim23p. By in vivo deletion and complementation analysis, we show that at least one extension is necessary for Aim23p function. At the same time, human mitochondrial IF3 is fully functional in yeast mitochondria even without both terminal extensions. While Escherichia coli IF3 itself is poorly active in yeast mitochondria, adding Aim23p terminal extensions makes the resulting chimeric protein as functional as the cognate factor. Our results show that the terminal extensions of IF3 have evolved as the "adaptors" that accommodate the translation factor of bacterial origin to the evolutionary changed protein biosynthesis system in mitochondria.

60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3.

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for "60S state" formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

A Moonlighting Human Protein Is Involved in Mitochondrial Import of tRNA.

In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).