Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Article (PDF Available)inNucleic Acids Research 41(1) · October 2012with55 Reads
DOI: 10.1093/nar/gks958 · Source: PubMed
Abstract
Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3's known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

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    • "Eukaryotic elongation factor 2 (eEF2) is a member of the GTPase superfamily of proteins which is known to assist in the tRNA translocation step of protein synthesis. Sordarin is a known antifungal agent which targets eEF2 so as to inhibit protein translation either by blocking eEF2-mediated translocation of tRNAs from A and P sites to P and E sites of the ribosome [4], or by interfering with eEF2-dependent ribosome splitting [5]. Interestingly, sordarin does not hinder GTP hydrolysis. "
    [Show abstract] [Hide abstract] ABSTRACT: Fungal infections have become a significant problem for immunosuppressed patients. Sordarin, a promising fungicidal agent, inhibits fungal protein synthesis by impairing elongation factor-2 (eEF2) function. Intriguingly, despite high sequence similarity among eEF2s from different species, sordarin has been shown to inhibit translation specifically in certain fungi while unable to do so in some other fungal species (e.g. Candida parapsilosis and Candida lusitaniae). The sordarin binding site on eEF2 as well as its mechanism of action is known. In a previous study, we have detailed the interactions between sordarin and eEF2 cavities from different fungal species at the molecular level and predicted the probable cause of sordarin sensitivity. Guided by our previous analysis, we aimed for computer-aided designing of sordarin derivatives as potential fungicidal agents that still remain ineffective against human eEF2. We have performed structural knowledge-based designing of several sordarin derivatives and evaluated predicted interactions of those derivatives with the sordarin-binding cavities of different eEF2s, against which sordarin shows no inhibitory action. Our analyses identify an amino-pyrrole derivative as a good template for further designing of promising broad-spectrum antifungal agents. The drug likeness and ADMET prediction on this derivative also supports its suitability as a drug candidate.
    Full-text · Article · Mar 2016
    • "However, some earlier studies also pointed out the recycling ability of some eukaryotic initiation factors like eIF3, eIF1, eIF3j [2]. Additionally, it has been proposed that eukaryotic elongation factors (eEF3, eEF2) have an ATP-dependent catalyzing role in the process of yeast post-termination ribosomal complex splitting [3,4]. The common aspect among all the recycling models discussed above is that all these are energy dependent (hydrolysis of ATP/ GTP). "
    [Show abstract] [Hide abstract] ABSTRACT: It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.
    Full-text · Article · Dec 2015
    • "Ribosome recycling represents the reaction to recycle the spent ribosome for the next round of translation of new mRNA. Kurata et al. [36] have recently reported that paromomycin negatively affects all different ribosome recycling steps to produce a dramatic effect on translation efficiency. Thus, increased resistance to paromomycin can be the result of enhanced translation efficiency due to an increased number of ribosomes available to a new round of mRNA translation. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Comparative transcriptomics and functional studies of different Saccharomyces species have opened up the possibility of studying and understanding new yeast abilities. This is the case of yeast adaptation to stress, in particular the cold stress response, which is especially relevant for the food industry. Since the species Saccharomyces kudriavzevii is adapted to grow at low temperatures, it has been suggested that it contains physiological adaptations that allow it to rapidly and efficiently acclimatise after cold shock. Results In this work, we aimed to provide new insights into the molecular basis determining this better cold adaptation of S. kudriavzevii strains. To this end, we have compared S. cerevisiae and S. kudriavzevii transcriptome after yeast adapted to cold shock. The results showed that both yeast mainly activated the genes related to translation machinery by comparing 12°C with 28°C, but the S. kudriavzevii response was stronger, showing an increased expression of dozens of genes involved in protein synthesis. This suggested enhanced translation efficiency at low temperatures, which was confirmed when we observed increased resistance to translation inhibitor paromomycin. Finally, 35S-methionine incorporation assays confirmed the increased S. kudriavzevii translation rate after cold shock. Conclusions This work confirms that S. kudriavzevii is able to grow at low temperatures, an interesting ability for different industrial applications. We propose that this adaptation is based on its enhanced ability to initiate a quick, efficient translation of crucial genes in cold adaptation among others, a mechanism that has been suggested for other microorganisms. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-432) contains supplementary material, which is available to authorized users.
    Full-text · Article · Jun 2014
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