Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.     

Purpuromycin: an antibiotic inhibiting tRNA aminoacylation.

Purpuromycin, an antibiotic produced by Actinoplanes ianthinogenes, had been reported previously to inhibit protein synthesis. In the present report, we demonstrate that the mechanism of action of this antibiotic is quite novel in that it binds with fairly high affinity to all tRNAs, inhibiting their acceptor capacity. Although more than one molecule of purpuromycin is bound to each tRNA molecule, the inhibitory activity of this antibiotic was found to be selective for the tRNA acceptor function; in fact, after the aminoacylation step, purpuromycin was found to affect none of the other tested functions of tRNA (interaction with the ribosomal P- and A-sites and interaction with translation factors). Accordingly, purpuromycin was found to inhibit protein synthesis only when translation depended on the aminoacylation of tRNA and not when the system was supplemented with pre-formed aminoacyl-tRNAs. Because purpuromycin did not interfere with the ATP-PPi exchange reaction of the synthetase or with the initial interaction of the enzyme with its tRNA substrate, the basis for the inhibition of aminoacylation is presumably the formation of a nonproductive synthetase-tRNA complex in the presence of purpuromycin in which the tRNA is unable to be charged with the corresponding amino acid.     

Various effects of paromomycin on tmRNA-directed trans-translation

trans-Translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. The results of a chemical footprinting study indicated that paromomycin molecules bind tmRNA at G332/G333 in the tRNA domain and A316 in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound at G332/G333 inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA-binding protein. It was also found that paromomycin causes a shift of the translation resuming point on tmRNA by -1. The effect on initiation shift was canceled by a mutation at the paromomycin-binding site in 16 S rRNA but not by mutations in tmRNA. A high concentration of paromomycin inhibited trans-translation, whereas it enhanced the initiation-shifted trans-translation when SmpB was exogenously added or a mutation was introduced at 333. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.     

Aptamer redesigned tRNA is nonfunctional and degraded in cells

An RNA aptamer derived from tRNA(Gln) isolated in vitro and a rationally redesigned tRNA(Gln) were used to address the relationship between structure and function of tRNA(Gln) aminoacylation in Escherichia coli. Two mutant tRNA(Gln) sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNA(Gln) in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNA(Gln) knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro.     

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.     

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys

Aminoacyl-tRNA synthetases(aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs). Previously, two mutations(c.1129 GA and c.517 TC) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNA~(Lys), with the c.517TC mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129GA and c.517TC mutations and provided yeast genetic bases to investigate other KARS mutations in the future.     

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.     

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.     

Yeast aspartyl-tRNA synthetase binds specifically its own mRNA

Dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast has a modular architecture and includes an N-terminal appendix of 70 amino acid residues that protrudes from the anticodon-binding module. This extension, of predicted helical structure, is not essential for aminoacylation but contains an RNA-binding motif that promotes non-specific interactions with tRNAs. As shown here, this protein extension can also interact with the 5' end of the AspRS mRNA. In vitro, optimal binding occurs on an mRNA domain comprising part of the 87 nucleotide long 5'UTR and the sequence encoding the N-terminal appendix. At the protein side, only the appendix and the anticodon-binding module participate in the interaction between AspRS and the mRNA domain. Binding is specific, since only tRNA(Asp) can dissociate the complex. In vivo, AspRS also binds specifically this mRNA domain and in doing so triggers a reduced translation of a fused GFP mRNA. From that, a mechanism for the regulation of this eukaryotic aminoacyl-tRNA synthetase is proposed. Implications for aspartylation accuracy in yeast are given.     

Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors.

The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.     

Methionyl-tRNA synthetase from Caenorhabditis elegans: a specific multidomain organization for convergent functional evolution

Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl-tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA-protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA-protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes.     

Interrelation between transfer RNA and amino-acid-activating sites of methionyl transfer RNA synthetase from Escherichia coli.

Binding of tRNA(Met/f) to the monomeric trypsin-modified methionyl-tRNA synthetase turns off the methionine-dependent isotopic ATP--PPi exchange. In the case of the dimeric native methionyltRNA synthetase, one anticooperatively bound tRNA(Met/f) inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3'-terminal adenosine. Esterification by methionine of the 3' end of tRNA reinforces the affinity of tRNA(Met/f)for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl-tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site. Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNA(Met/f).