Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells

Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it induced import of endogenous cytosolic tRNAs, including tRNA(Lys), and restored mitochondrial function in a cybrid harboring a mutant mt tRNA(Lys) (MT-TK) gene. The use of protein complexes to modulate mitochondrial function may help in the management of such genetic disorders.     

Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

Accuracy of in vivo aminoacylation requires proper balance of tRNA and aminoacyl-tRNA synthetase

The fidelity of protein biosynthesis in any cell rests on the accuracy of aminoacylation of tRNA. The exquisite specificity of this reaction is critically dependent on the correct recognition of tRNA by aminoacyl-tRNA synthetases. It is shown here that the relative concentrations of a tRNA and its cognate aminoacyl-tRNA synthetase are normally well balanced and crucial for maintenance of accurate aminoacylation. When Escherichia coli Gln-tRNA synthetase is overproduced in vivo, it incorrectly acylates the supF amber suppressor tRNA(Tyr) with Gln. This effect is abolished when the intracellular concentration of the cognate tRNA(Gln2) is also elevate. These data indicate that the presence of aminoacyl-tRNA synthetase and the cognate tRNAs in complexed form, which requires the proper balance of the two macromolecules, is critical in maintaining the fidelity of protein biosynthesis. Thus, limits exist on the relative levels of tRNAs and aminoacyl-tRNA synthetases within a cell.     

Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase

An analysis of the aminoacylation kinetics of unmodified yeast tRNAPhe mutants revealed that five single-stranded nucleotides are important for its recognition by yeast phenylalanyl-tRNA synthetase, provided they were positioned correctly in a properly folded tRNA structure. When four other tRNAs were changed to have these five nucleotides, they became near-normal substrates for the enzyme.     

RNA required for import of precursor proteins into mitochondria

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.     

Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation

Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis. Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body. The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated. Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.     

An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.     

Method for Isolating Mitochondrial DNA from Etiolated Tissue of Cabbage

Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.     

Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission

In sexual reproduction of most animals, the spermatozoon provides DNA and centrioles, together with some cytoplasm and organelles, to the oocyte that is being fertilized. Paternal mitochondria and their genomes are generally eliminated in the embryo by an unknown degradation mechanism. We show that, upon fertilization, a Caenorhabditis elegans spermatozoon triggers the recruitment of autophagosomes within minutes and subsequent paternal mitochondria degradation. Whereas the nematode-specific sperm membranous organelles are ubiquitinated before autophagosome formation, the mitochondria are not. The degradation of both paternal structures and mitochondrial DNA requires an LC3-dependent autophagy. Analysis of fertilized mouse embryos shows the localization of autophagy markers, which suggests that this autophagy event is evolutionarily conserved to prevent both the transmission of paternal mitochondrial DNA to the offspring and the establishment of heteroplasmy.     

Protein sorting to mitochondria: evolutionary conservations of folding and assembly

According to the endosymbiont hypothesis, mitochondria have lost the autonomy of their prokaryotic ancestors. They have to import most of their proteins from the cytosol because the mitochondrial genome codes for only a small percentage of the polypeptides that reside in the organelle. Recent findings show that the sorting of proteins into the mitochondrial subcompartments and their folding and assembly follow principles already developed in prokaryotes. The components involved may have structural and functional equivalents in bacteria.     

Association of transfer RNA acceptor identity with a helical irregularity

The aminoacylation specificity ("acceptor identity") of transfer RNAs (tRNAs) has previously been associated with the position of particular nucleotides, as opposed to distinctive elements of three-dimensional structure. The contribution of a G.U wobble pair in the acceptor helix of tRNA(Ala) to acceptor identity was examined with synthetic amber suppressor tRNAs in Escherichia coli. The acceptor identity was not affected by replacing the G.U wobble pair in tRNA(Ala) with a G.A, C.A, or U.U wobble pair. Furthermore, a tRNA(Ala) acceptor identity was conferred on tRNA(Lys) when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNA(Ala) show that a substantial acceptor identity was retained when the G.U wobble pair was translocated to another site in the acceptor helix. These results suggest that the G.U wobble pair induces an irregularity in the acceptor helix of tRNA(Ala) to match a complementary structure in the aminoacylating enzyme.     

禾谷类作物编码与雄性不育相关线粒体蛋白核基因筛选策略

植物细胞质雄性不育（CMS）是指植物不能产生具有正常功能的花粉。控制CMS的基因存在于线粒体中,核基因对线粒体基因表达具顺向调节作用,而线粒体对核基因表达具有反向调节作用。目前植物线粒体蛋白组研究主要以营养器官为材料,主要粮食作物如水稻、小麦及玉米等都存在花药小、花期短、同一花序的花药发育不同步等。文章介绍了线粒体起源及植物线粒体蛋白组和植物CMS的研究现状,禾谷类作物花药线粒体蛋白组研究的重要性及困难,提出利用比较基因组学筛选水稻编码线粒体蛋白、与雄性不育相关的核基因的策略,以期为研究禾谷类作物特别是水稻CMS机理提供参考。     

Mitochondrial transformation in yeast by bombardment with microprojectiles

The genetic transformation of mitochondria and chloroplasts has been an intractable problem. The newly developed "biolistic" (biological ballistic) process was used to deliver DNA into yeast cells to stably transform their mitochondria. A nonreverting strain, which is respiratory deficient because of a deletion in the mitochondrial oxi3 gene, was bombarded with tungsten microprojectiles coated with DNA bearing sequences that could correct the oxi3 deletion. Respiratory-competent transformants were obtained in which the introduced oxi3 DNA is integrated at the homologous site in the mitochondrial genome. Organelle genomes can now be manipulated by molecular genetic techniques in the same way as nuclear genomes.     

ER tubules mark sites of mitochondrial division

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.     

Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.