Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.     

Arginine changes the conformation of the arginine attenuator peptide relative to the ribosome tunnel

The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.     

The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.     

A structural model for the large subunit of the mammalian mitochondrial ribosome

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.     

The mitochondrial genome of Saprolegnia ferax: organization, gene content and nucleotide sequence

The mitochondrial genome of the peronosporomycete water mold Saprolegnia ferax has been characterized as a 46 930 bp circle containing an 8618 bp large inverted repeat (LIR). Eighteen reading frames encode identified subunits of respiratory complexes I, III, IV and V; 16 encode polypeptides of small and large mitoribosome subunits; and one encodes a subunit of the sec-independent protein translocation pathway. Of four additional putative reading frames three are homologues of those found in the related Phytophthora infestans genome. Protein encoding loci in the tightly compacted genome typically are arranged in operon-like clusters including three abutting and two overlapping pairs of reading frames. Translational RNAs include the mitochondrial small and large subunit rRNAs and 25 tRNA species. No tRNAs are encoded to enable translation of any threonine or the arginine CGR codons. The LIR separates the molecule into 19 274 bp large and 10 420 bp small single copy regions, and it encodes intact duplicate copies of four reading frames encoding known proteins, both rRNAs, and five tRNAs. Partial 3' sequences of three additional reading frames are duplicated at single copy sequence junctions. Active recombination between LIR elements generates two distinctive gene orders and uses the duplicated 3' sequences to maintain intact copies of the partially duplicated loci.     

The Effects of Canavanine on Protein and Nucleic Acid Synthesis in Canavanine Resistant and Sensitive Species of Higher Plants

The effectiveness of the inhibitor, canavanine, was evaluated by examining its action in Canavalia ensiformis and Glycine max. Isolated roots were grown in culture tubes containing White's medium plus canavanine or arginine. A differential effect of canavanine on the incorporation of precursors of DNA, RNA, and protein was found, which is assumed to be related to the ability of the plant to utilize canavanine in reactions typically involving arginine. Canavanine was not found to affect DNA, RNA, or protein synthesis in Canavalia ensiformis, a plant in which this amino acid is synthesized naturally. In the canavanine sensitive species, Glycine max, of the same subfamily Papilionoideae, canavanine was observed to inhibit strongly DNA, RNA, and protein synthesis. A primary inhibition of the RNA synthesizing system is suggested. The data indicate the canavanine inhibitions are more complex than a simple competition with arginine in protein synthesis.     

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.     

Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.     

Nascent polypeptide sequences that influence ribosome function

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.     

Canavanine incorporation into the antibacterial proteins of the fly, Phormia terranovae (Diptera), and its effect on biological activity

In response to microbial infection or mechanical injury, larvae of the fly, Phormia terranovae (Diptera), can induce de novo production of a group of antibacterial proteins including: peak I protein, diptericin A, diptericin B, diptericin C, and peak V protein. Administration of L-canavanine at the time of mechanical injury results in the incorporation of this arginine antagonist into these proteins. Canavanine replacement for arginine causes a total loss of detectable antibacterial activity for diptericin B and diptericin C, whereas diptericin A and peak V protein are severely inhibited. This loss in biological activity occurs in spite of the fact that canavanine stimulates induced protein synthesis. Analysis of the hydrolysate of diptericin A reveals that one-third of the 3 arginyl residues are replaced by canavanine. This investigation provides the first evidence that canavanine incorporation into a protein can impair its function.     

Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome. Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria

The mammalian mitochondrial ribosome (mitoribosome) is a highly protein-rich particle in which almost half of the rRNA contained in the bacterial ribosome is replaced with proteins. It is known that mitochondrial translation factors can function on both mitochondrial and Escherichia coli ribosomes, indicating that protein components in the mitoribosome compensate the reduced rRNA chain to make a bacteria-type ribosome. To elucidate the molecular basis of this compensation, we analyzed bovine mitoribosomal large subunit proteins; 31 proteins were identified including 15 newly identified proteins with their cDNA sequences from human and mouse. The results showed that the proteins with binding sites on rRNA shortened or lost in the mitoribosome were enlarged when compared with the E. coli counterparts; this suggests the structural compensation of the rRNA deficit by the enlarged proteins in the mitoribosome.     

arg—13可能参与鸟氨酸在粗糙脉孢霉线粒体的过膜转运

arg-13 is a leaky mutation involved in arginine metabolism. A tight selection is developed using similar amount of lysine and ornithine replacing other nitrogen source in minimal medium. This selection strongly inhibits the growth of arg-13 under stringent sorbose/glucose condition but allows arg-13 to grow under spot test conditions. As ornithine is build up through mitochondrial ornithine biosynthesis and transport from cytoplasm to mitochondria, arg-13 is combined in genetic crosses with arg-4 which blocks mitochondrial ornithine synthesis. Under spot test conditions, double mutant arg-4, arg-13 is able to use ornithine as sole nitrogen source and arginine biosynthesis precursor, but subject to strong lysine and canavanine inhibition. While the usage of ornithine in arg-4 single mutant with intact ornithine transport function is only slightly inhibited by lysine. All available data suggest arg-13 plays a major role in mitochondrial ornithine transport. The strain carrying the mutation at the arg-13 locus allows inefficient mitochondrial ornithine trafficking, possibly mediated by another distinct basic amino acid carrier.     

Mitochondrial protein synthesis: Figuring the fundamentals,complexities and complications,of mammalian mitochondrial translation

Mitochondrial protein synthesis is essential for all mammals, being responsible for providing key components of the oxidative phosphorylation complexes. Although only thirteen different polypeptides are made, the molecular details of this deceptively simple process remain incomplete. Central to this process is a non-canonical ribosome, the mitoribosome, which has evolved to address its unique mandate. In this review, we integrate the current understanding of the molecular aspects of mitochondrial translation with recent advances in structural biology. We identify numerous key questions that we will need to answer if we are to increase our knowledge of the molecular mechanisms underlying mitochondrial protein synthesis.     

Site of synthesis of the proteins of mammalian mitochondrial ribosomes. Evidence from cultured bovine cells

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeling in the absence of cytoplasmic protein synthesis produced a "blank" fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [35S]methionine into the enumerated mitoribosomal proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

The polypeptide tunnel exit of the mitochondrial ribosome is tailored to meet the specific requirements of the organelle

The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co‐translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co‐factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially‐encoded proteins.     

Protein tagging at rare codons is caused by tmRNA action at the 3' end of nonstop mRNA generated in response to ribosome stalling

It has been believed that protein tagging caused by consecutive rare codons involves tmRNA action at the internal mRNA site. We demonstrated previously that ribosome stalling either at sense or stop codons caused by certain arrest sequences could induce mRNA cleavage near the arrest site, resulting in nonstop mRNAs that are recognized by tmRNA. These findings prompted us to re-examine the mechanism of tmRNA tagging at a run of rare codons. We report here that either AGG or CGA but not AGA arginine rare-codon clusters inserted into a model crp mRNA encoding cAMP receptor protein (CRP) could cause an efficient protein tagging. We demonstrate that more than three consecutive AGG codons are needed to induce an efficient ribosome stalling therefore tmRNA tagging in our system. The tmRNA tagging was eliminated by overproduction of tRNAs corresponding to rare codons, indicating that a scarcity of the corresponding tRNA caused by the rare-codon cluster is an important factor for tmRNA tagging. Mass spectrometry analyses of proteins generated in cells lacking or possessing tmRNA encoding a protease-resistant tag sequence indicated that the truncation and tmRNA tagging occur within the cluster of rare codons. Northern and S1 analyses demonstrated that nonstop mRNAs truncated within the rare-codon clusters are detected in cells lacking tmRNA but not in cells expressing tmRNA. We conclude that a ribosome stalled by the rare codon induces mRNA cleavage, resulting in nonstop mRNAs that are recognized by tmRNA.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.