<p>Becoming antibiotic resistant is often associated with fitness costs for the resistant bacteria. This is seen as a loss of competitiveness against the antibiotic-sensitive wild-type in an antibiotic-free environment. In this study, the physiological alterations associated with fitness cost of antibiotic resistance <i>in vitro</i> (in the laboratory medium), and <i>in vivo</i> (in a mouse infection model), are identified in the model system of fusidic acid resistant (Fus^R) <i>Salmonella</i> <i>enterica</i> serovar Typhimurium.</p><p>Fus^R mutants have mutations in <i>fusA</i>, the gene that encodes translation elongation factor G (EF-G). Fus^R EF-G has a slow rate of regeneration of active EF-G·GTP off the ribosome, resulting in a slow rate of protein synthesis. The low fitness of Fus^R mutants <i>in vitro</i>, and <i>in vivo</i>, can be explained in part by a slow rate of protein synthesis and resulting slow growth. However, some Fus^R mutants with normal rates of protein synthesis still suffer from reduced fitness <i>in vivo</i>. We observed that Fus^R mutants have perturbed levels of the global regulatory molecule ppGpp. One consequence of this is an inefficient induction of RpoS, a regulator of general stress reponse and an important virulence factor for <i>Salmonella</i>. In addition, we found that Fus^R mutants have reduced amounts of heme, a co-factor of catalases and cytochromes. As a consequence of the heme defect, Fus^R mutants have a reduced ability to withstand oxidative stress and a low rate of aerobic respiration.</p><p>The pleiotropic phenotypes of Fus^R mutants suggest that antibiotic resistance can be associated with broad changes in bacterial physiology. Knowledge of physiological alterations that reduce the fitness of antibiotic-resistant mutants can be useful in identifying novel targets for antimicrobial agents. Drugs that alter the levels of global transcriptional regulators such as ppGpp or RpoS deserve attention as potential antimicrobial agents. Finally, the observation that Fus^R mutants have increased sensitivity to several unrelated classes of antibiotics suggests that the identification of physiological cost of resistance can help in optimizing treatment of resistant bacterial populations.</p>

<p>Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. </p><p>In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. </p><p>The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. </p><p>In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.</p>

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.

Becoming antibiotic resistant is often associated with fitness costs for the resistant bacteria. This is seen as a loss of competitiveness against the antibiotic-sensitive wild-type in an antibiotic-free environment. In this study, the physiological alterations associated with fitness cost of antibiotic resistance in vitro (in the laboratory medium), and in vivo (in a mouse infection model), are identified in the model system of fusidic acid resistant (FusR) Salmonella enterica serovar Typhimurium. FusR mutants have mutations in fusA, the gene that encodes translation elongation factor G (EF-G). FusR EF-G has a slow rate of regeneration of active EF-G·GTP off the ribosome, resulting in a slow rate of protein synthesis. The low fitness of FusR mutants in vitro, and in vivo, can be explained in part by a slow rate of protein synthesis and resulting slow growth. However, some FusR mutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. We observed that FusR mutants have perturbed levels of the global regulatory molecule ppGpp. One consequence of this is an inefficient induction of RpoS, a regulator of general stress reponse and an important virulence factor for Salmonella. In addition, we found that FusR mutants have reduced amounts of heme, a co-factor of catalases and cytochromes. As a consequence of the heme defect, FusR mutants have a reduced ability to withstand oxidative stress and a low rate of aerobic respiration. The pleiotropic phenotypes of FusR mutants suggest that antibiotic resistance can be associated with broad changes in bacterial physiology. Knowledge of physiological alterations that reduce the fitness of antibiotic-resistant mutants can be useful in identifying novel targets for antimicrobial agents. Drugs that alter the levels of global transcriptional regulators such as ppGpp or RpoS deserve attention as potential antimicrobial agents. Finally, the observation that FusR mutants have increased sensitivity to several unrelated classes of antibiotics suggests that the identification of physiological cost of resistance can help in optimizing treatment of resistant bacterial populations.

Cannabinoids have been shown to alter synaptic plasticity but the mechanism by which this occurs at hippocampal CA3-CA1 synapses in vivo is not yet known. Utilizing in vivo electrophysiological recordings of field excitatory postsynaptic potentials (fEPSP) on anesthetized rats and mice as well as three lines of conditional knockout mouse models, the objective was to show a two-part mechanistic breakdown of cannabinoid-evoked CA3-CA1 long-term depression (LTD) in its induction as well as early and later-phase expression stages. It was determined that this cannabinoid-induced in vivo LTD requires cannabinoid type-1 receptors (CB1Rs) on astrocytes, but not CB1Rs on glutamatergic or GABAergic neuronal axons/terminals. Pharmacological testing determined that cannabinoid-induced in vivo LTD also requires activation of NMDA receptors (NMDAR) and subsequent postsynaptic endocytosis of AMPA receptors (AMPAR). There exists a clear role for NR2B-containing NMDARs in a persistent, transitory form, potentially related to prolonged or delayed glutamate release (possibly as a result of the astrocytic network). A key determination of the expression phase is the involvement of new protein synthesis (using translation and transcription inhibitors) – further evidence of the long-term action of the synaptic plasticity from a single cannabinoid dose.

Long-term potentiation (LTP) and depression (LTD) are important forms of activity-dependent synaptic plasticity believed to play a role in memory at the cellular level. It has previously been described that synthesis of new proteins is needed to maintain LTP longer than a few hours. Other reports argue that sufficient proteins for stable LTP are already available. The present study aims to examine the role of protein synthesis in LTD, the presumed mirror mechanism of LTP. Experiments were carried out in hippocampal slices from young (12-45 days) and old (12-18 weeks) Sprague-Dawley rats. Extracellular techniques were used to study synaptic responses in the Schaffer-collateral-commissural pathway. Plasticity was induced electrically by low frequency stimulation (2-3 trains at 1 Hz for 15 min) or chemically by brief exposure to certain glutamate receptor agonists (NMDA at 20 µM for 3 min or DHPG at 100 µM for 10 min). Whole slice protein synthesis was quantified by assessing 3H-leucine incorporation. Stable LTD (&gt; 8 h) was be obtained by either electrical or chemical activation. Protein synthesis inhibitors anisomycin (40 uM) and cycloheximide (100 uM) both failed to influence the magnitude of LTD. Moreover, no age difference was found, in terms of stable LTD in both young and old rats under inhibition of protein synthesis. The potency of the inhibitors was found to be high, depressing synthesis down to a few percent. It is concluded that sufficient proteins for generating stable LTD are normally present in the brain, implying a large safety-margin for cellular memory.

This thesis entitled “Mechanism of Anticancer and Antimalarial Action of a Modulator of Heat Shock Proteins” describes the successful elucidation of the mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG). DSG, a relatively well known immunosuppressant and antitumor molecule has been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90, enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998), and modulates several seemingly unrelated cellular processes. DSG has also been demonstrated to inhibit protein synthesis and polyamine synthesis in cells (Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit malaria parasite growth by inhibiting polyamine synthesis. The grim situation with regard to malaria infection and mortality, principally an offshoot of the emergence of chloroquine resistant strains of the causative agent of malaria - Plasmodium falciparum, calls for intense efforts towards developing efficacious antimalarial agents with few side effects. DSG, having been used already in graft rejection cases in man and demonstrated to potently inhibit malaria in mice (Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of interest to solve the mystery of its mechanism of antimalarial action. Chapter 1 surveys literature related to DSG mechanism of action and presents the thesis objective. Chapter 1 also gives an overview of heat shock proteins and their role in cancer, and the biology of the malaria parasite (Plasmodium falciparum), the working of the principal metabolic pathways existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast processes are dealt with in more detail. Though DSG has been speculated to kill the malaria parasite by inhibiting polyamine synthesis, that DSG could clear malaria infection in Plasmodium berghei infected mice did not corroborate with the observation that inhibitors of polyamine biosynthesis are incapable of inhibiting the malaria parasite in vivo probably because the parasites make do with polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On the other hand, DSG is known to bind heat shock proteins, and inhibit protein synthesis, and heat shock proteins are speculated to be involved in the activation of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It seemed likely that if HRI is activated upon sequestration of heat shock proteins by DSG, it would culminate in protein synthesis inhibition and ultimately, cell death. With the intention to investigate this line of thought, the PlasmodB database was mined for proteins essential to the existence of heme dependent protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated in DSG binding, and one without, and an Hsp70 interacting protein were cloned and expressed in their recombinant form in Escherichia coli. The preliminary characterization of these heat shock proteins described in Chapter 2 revealed that they were functionally active. DSG did not inhibit either the chaperone activity of the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase activity as anticipated. Chapter 3 gives a complete picture of the mechanism of protein synthesis inhibition by DSG in the standard protein synthesis system – reticulocyte lysate. The experiments carried out revealed that DSG inhibits protein synthesis precisely through the mechanism envisaged, i.e. through phosphorylation of HRI following sequestration of Hsp70. Experiments involving exogenous addition of heat shock protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell lines inversely correlated with the inhibitory activity of DSG, and modulation of Hsp70 levels through standard methods altered DSG inhibition of protein synthesis in these cells. It was thus confirmed that DSG did indeed inhibit mammalian cells through the pathway envisaged. Its previously reported antitumor property is probably through this outlined mechanism of interference with protein regulation. In the malaria parasite, too, DSG inhibited protein synthesis through eIF2 alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4. However, while the concentration of DSG required for inhibition of malaria parasite growth was in the nanomolar range, high micromolar concentrations of DSG were required to effect protein synthesis inhibition in the malaria parasite, indicating that yet another target for DSG existed in the malaria parasite. With protein synthesis no longer a candidate target of DSG, I looked into the previously implicated polyamine synthesis pathway. In the event of DSG inhibiting polyamine transport in addition to polyamine biosynthesis, it would be expected to clear malaria infection in vivo contrary to other inhibitors of polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis pathway in the malaria parasite is provided. Experiments involving incorporation of radiolabeled precursors in the malaria parasite and in mammalian cells, however, revealed that only high micromolar concentrations of DSG inhibit polyamine synthesis. Polyamine transport was also studied in considerable detail in malaria parasite infected red blood cells. Though infected red blood cells demonstrated different kinetic parameters, implying that new polyamine transporters were employed by the parasite on the red blood cell upon infection, DSG did not potently inhibit polyamine transport, either. The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997). DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis – processes that occur in the apicoplast, but effected a decrease in the amount of nucleus encoded proteins that are targeted to the apicoplast, suggesting that it inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal microscopy of parasites transfected with GFP fusion protein confirmed these findings, and is described in Chapter 6. The thesis ends with a summary of the findings in Chapter 7. Apicoplast processes have always been considered to harbor immense potential in the development of antimalarial agents, thanks to the absence of an equivalent organelle and hence pathways, in the human host. Trafficking of nucleus encoded proteins to the apicoplast has remained unexplored however. The work done in this thesis not only serves to demystify DSG with regard to its mechanism of action, but also paves the way for further studies in this area of intracellular trafficking, which could help in the development of more efficacious antimalarial agents. It also adds a new dimension to previous work conducted with regard to the anticancer action of DSG. Appendix 1 revolves around inhibitors which target various apicoplast processes. Apicoplast processes have been conventionally linked to the intriguing but unfortunate (with respect to clinical application) “delayed death”. Results presented in this section demonstrate that not all apicoplast processes invoke “delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis and heme synthesis evoke rapid death. Inhibitors designed to target these processes could, therefore, be highly efficacious.

The studies reported in this thesis address, firstly, aspects of ribosome recycling in eubacteria, and secondly, a preliminary characterization of an EFG-like locus from Mycobacterium smegmatis. A hitherto unsuspected role of the ribosome recycling factor in governing the fidelity of initiation has been discovered during the course of this work. A summary of the relevant literature is presented in chapter 1. Section I of the ‘General Introduction’ provides a brief review of the current understanding of protein biosynthesis, with a special emphasis on ribosome recycling and the fidelity of translation initiation. Section II provides a brief introduction to mycobacterial translation, and known deviations from the E. coli prototype are highlighted. This is followed by three chapters containing experimental work, as summarized below. (i) Role of elongation factor G in governing specificity of ribosome recycling In eubacteria and the eukaryotic organelles, the post-termination ribosome complexes are recycled by the combined action of ribosome recycling factor (RRF) and elongation factor G (EFG). Earlier studies both from our laboratory and other laboratories have revealed the existence of specific interactions between RRF and EFG that are crucial for ribosome recycling, using ribosomes from E. coli and factors from both E. coli and heterologous sources such as Mycobacterium tuberculosis, Thermus thermophilus etc. In this study, to further understand the mechanism of ribosome recycling, we employed polysomes from both E. coli and M. smegmatis and monitored ribosome recycling in in vitro assays using RRF and EFG from both these sources; in addition, in vivo assays were performed in E. coli using either temperature-sensitive strains or strains carrying a deletion in frr (encoding RRF) or fusA (encoding EFG) genes. It was found that, in E. coli, RRF from Mycobacterium tuberculosis and M. smegmatis function with MtuEFG or MsmEFG but not with EcoEFG. In vitro assays revealed that the mycobacterial EFGs facilitate recycling of both the mycobacterial and E. coli polysomes not only with mycobacterial RRFs but also with EcoRRF. In contrast, although EcoEFG binds to mycobacterial polysomes, carries out GTP hydrolysis and is reported to sustain translocation on mycobacterial ribosomes, its activity in recycling mycobacterial polysomes was undetectable with EcoRRF, as well as with the mycobacterial RRFs. Such an observation allowed us to infer that EFG establishes specific interactions with the ribosome that are crucial for ribosome recycling but not for translocation, suggesting that translocation and ribosome recycling are distinct functions of EFG. In addition, a number of EFG chimeras generated by swapping corresponding domains between Msm- and Eco-EFGs were analyzed for their ability to sustain translocation and/or ribosome recycling in E. coli and M. smegmatis, using a combination of in vivo (for E. coli) and in vitro (for both E. coli and M. smegmatis) approaches. Our observations reveal that a dual set of specific interactions of EFG with RRF and ribosome is essential for ribosome recycling. While the RRF-EFG specific interactions are predominantly localized to the domains IV and V of EFG, the EFG-ribosome specific interactions that are crucial for ribosome recycling are not localized to a specific region of EFG but are found throughout the molecule. Our novel observations also emphasize the importance of using ribosomes from heterologous sources to understand the mechanism of this crucial process. (ii) Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions; however, very little is known about the role of these rRNA modifications in ribosome function. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of the intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. The isolation of a folD122 mutant strain of E. coli with a deficiency in rRNA methylations, as well as the availability of E. coli strains deficient for various individual methyltransferases that modify specific rRNA residues, provided us with a genetic tool to assay the role of rRNA methylations in ribosome recycling. We observed that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, a compromise in the RRF activity was found to afford increased initiation with a mutant tRNAfMet wherein the three consecutive G-C base pairs (29GGG31:39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNAMet (29UCA31:39ψGA41). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. In addition, it was also found that IF3 and rRNA methylations, both of which are known to affect fidelity of initiation, exert their effects through distinct mechanisms, despite the proximity of a cluster of methylated rRNA residues to the IF3 binding site on the 30S subunit. (iii) Characterization of the role of EFG2, an EFG-like locus in Mycobacterium smegmatis Several bacteria, including various species of mycobacteria (with the exception of Mycobacterium leprae) contain a second EFG-like locus, denoted as fusA2, which shows considerable homology to fusA (encoding EFG). A comparison of the sequences of EFG and EFG2 from various bacteria reveals that EFG2 contains a GTPase domain and domains with significant homology to EFG domains IV and V, suggesting that it may function as an elongation factor. With the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like protein factors has not been studied so far. Hence, it was of interest to characterize EFG2. In the current study, EFG2 from M. smegmatis was characterized both by in vitro biochemical assays as well as by in vivo experiments targeted to investigate the biological significance of EFG2 in mycobacteria. It was found that, unlike EFG, MsmEFG2 could not sustain either translocation or ribosome recycling in E. coli. Despite the fact that the purified MsmEFG2 could bind guanine nucleotides, it lacked the ribosome-dependent GTPase activity characteristic of EFG and other translation GTPases, suggesting that it was unlikely to function as an elongation factor. However, EFG2 was found to be expressed in stationary phase cultures of M. smegmatis. To understand the biological significance of EFG2, fusA2 was disrupted in M. smegmatis. The viability of the M. smegmatis mc2155 fusA2::kan derivative indicates that MsmfusA2 is a non-essential gene. While disruption of the fusA2 gene (encoding EFG2) in M. smegmatis does not appear to affect its growth and survival in log phase or stationary phase or under hypoxic conditions, preliminary experiments indicate that disruption of fusA2 confers a fitness disadvantage to M. smegmatis when competed against M. smegmatis mc2155 (with wild type fusA2 locus).

Στην παρούσα διατριβή μελετήθηκε ο ρόλος της έλικας h44 του 18S rRNA του Saccharomyces cerevisiae επί διαφόρων παραμέτρων της πρωτεϊνικής σύνθεσης. Η μελέτη διεξήχθη με την βοήθεια των σημειακών μεταλλάξεων A1491G (rdn15) και U1495C (rdnhyg1), οι οποίες εντοπίζονται στην Α-θέση του ριβοσώματος. Η μετάλλαξη rdn15 επιδρά ήπια στον ρυθμό ανάπτυξης των κυττάρων ενώ τα rdn15 ριβοσώματα επιτελούν την πρωτεϊνοσύνθεση με αυξημένη ακρίβεια. Η έλλειψη σοβαρών επιπτώσεων παρουσία της rdn15 φανερώνει ότι το νουκλεοτίδιο 1491 δεν παίζει καθοριστικό ρόλο στην λειτουργία του ριβοσώματος. Τα κύτταρα ζύμης που φέρουν την μετάλλαξη rdnhyg1 αναπτύσσονται βραδύτερα από τα κύτταρα αγρίου τύπου, ενώ τα rdnhyg1 ριβοσώματα πρωτεϊνοσυνθέτουν με ελαφρώς αυξημένη συχνότητα λάθους. Η μετάλλαξη αυξάνει επίσης την συγγένεια της Α-θέσης του ριβοσώματος για το αμινοακυλο-tRNA και επιδρά αρνητικά στο στάδιο της μετατόπισης, χωρίς να επηρεάζει την ενεργότητα πεπτιδυλοτρανσφεράσης. Η επίδραση της μετάλλαξης rdnhyg1 επί διαφόρων παραμέτρων της πρωτεϊνοσύνθεσης δικαιολογεί την συντήρηση της U1495 κατά την εξέλιξη. Η μετάλλαξη sup45-R2ts εντοπίζεται στο γονίδιο που κωδικοποιεί τον παράγοντα τερματισμού eRF1 και οδηγεί στην αντικατάσταση της προλίνης 86 από αλανίνη. Η μετάλλαξη δεν επηρεάζει τις περισσότερες από τις λειτουργίες του ριβοσώματος που εξετάστηκαν, αλλά μειώνει την μεταφραστική πιστότητα. Σε κύτταρα που φέρουν ταυτόχρονα την μετάλλαξη sup45-R2ts και την ριβοσωματική μετάλλαξη rdn15, η συχνότητα λάθους αυξάνεται σε βαθμό μεγαλύτερο από την αθροιστική επίδραση των δύο επιμέρους μεταλλάξεων, επιβεβαιώνοντας μια ιδιαίτερη αλληλεπίδραση του μεταλλαγμένου παράγοντα eRF1 με τα rdn15 ριβοσώματα, που, όπως προκύπτει, αντιστρέφει τον υπερακριβή χαρακτήρα των μεταλλαγμένων ριβοσωμάτων. Όταν η μετάλλαξη sup45-R2ts συνυπάρχει με την ριβοσωματική μετάλλαξη rdnhyg1 η συχνότητα λάθους δεν επηρεάζεται σημαντικά. Η rdnhyg1 φαίνεται να ελαχιστοποιεί την επίδραση του μεταλλαγμένου παράγοντα eRF1 ενισχύοντας την δράση GTPάσης του eRF3. Τα παραπάνω αποτελέσματα φανερώνουν επιπλέον ότι η sup45 δύναται να μεταβάλει τις ιδιότητας ορισμένων μεταλλάξεων κατά την ριβοσωματική λειτουργία. Το στέλεχος που φέρει την μετάλλαξη rdn15 είναι πολύ ευαίσθητο έναντι της παρομομυκίνης αλλά και έναντι της τομπραμυκίνης, αν και σε μικρότερο βαθμό. Τα αποτελέσματα αυτά αποδίδονται στην ικανότητα της μετάλλαξης να αυξάνει την συγγένεια της Α-θέσης του ριβοσώματος για τα εν λόγω αντιβιοτικά. Αντίθετα, η μετάλλαξη rdn15 προσδίδει ανθεκτικότητα στην υγρομυκίνη, φανερώνοντας ότι ο τρόπος πρόσδεσης και δράσης του συγκεκριμένου αμινογλυκοζίτη διαφοροποιείται. Το στέλεχος που φέρει την μετάλλαξη rdnhyg1 είναι ανθεκτικό και στα τρία αντιβιοτικά σε σύγκριση με το αγρίου τύπου, φανερώνοντας ότι η U1495 είναι καθοριστική για την πρόσδεση των αμινογλυκοζιτών στο ριβόσωμα. Τα κύτταρα που φέρουν την εξωριβοσωματική μετάλλαξη sup45-R2ts είναι πιο ευαίσθητα από τα αντίστοιχα αγρίου τύπου έναντι και των τριών αμινογλυκοζιτών. Ωστόσο η μετάλλαξη sup45-R2ts, δεν επηρεάζει την ικανότητα των αντιβιοτικών αυτών να προσδένονται στα αγρίου τύπου και μεταλλαγμένα ριβοσώματα και να επάγουν άμεσα τα μεταφραστικά λάθη. Η μελέτη επίδρασης των αμινγλυκοζιτών επιβεβαίωσε ότι η παρομομυκίνη και η υγρομυκίνη αναστέλλουν την ανάπτυξη των κυττάρων ζύμης, ενώ η τομπραμυκίνη δεν έχει καμία επίδραση. Το γεγονός αυτό συνδυάζεται με την αδυναμία της τομπραμυκίνης να αναστείλει την πρόσδεση του υποστρώματος στην Α-θέση των ριβοσωμάτων. Ωστόσο η τομπραμυκίνη, όπως η παρομομυκίνη και η υγρομυκίνη, είναι ικανή να αυξήσει την συχνότητα λάθους και την σύνθεση πολυφαινυλαλανίνης. / In present study, we investigated the role of helix h44 of 18S rRNA of Saccharomyces cerevisiae on several parameters of protein synthesis. For this purpose we employed mutations A1491G (rdn15) and U1495C (rdnhyg1) which are located in the A-site of the ribosome. The rdn15 mutation slightly delays cell growth, while rdn15 ribosomes translate proteins with higher fidelity. The lack of severe impairment of ribosomal function by mutation rdn15 indicates that the nature of nucleotide 1491 is not essential for ribosomal function. Yeast cells carrying the rdnhyg1 mutation grow slower than wild-type cells, while their ribosomes possess a slightly increased error rate. This mutation also increases the affinity of the A-site for aminoacyl-tRNA and renders ribosomes less efficient in translocation without affecting peptidyltransferase activity. The effect of mutation rdnhyg1 on several parameters of protein synthesis explains why U1495 is evolutionarily conserved. Mutation sup45-R2ts is located in the gene encoding eukaryotic Release Factor 1 (eRF1) and results in the substitution of proline 86 by alanine. This mutation leaves unaffected most ribosomal functions but it decreases translational fidelity. When ribosomal mutation rdn15 is introduced in cells already carrying sup45-R2ts mutation, the error frequency is increased to a degree which is higher than the additive effect of the two mutations, testifying to a previously reported special interaction of eRF1 with rdn15 ribosomes, which in this case reverses the hyperaccurate character of rdn15 ribosomes. When mutation sup45-R2ts is expressed in cells also carrying ribosomal mutation rdnhyg1, the error frequency is not significantly altered. Mutation rdnhyg1 seems to minimize the effect of the mutant factor eRF1 on ribosomal function by enhancing GTPase activity of eRF3. The results obtained with rdn15 and rdnhyg1 alone or in combination with sup45-R2ts show for the first time that the presence of sup45 may result in significant changes in the properties of the mutations under study. The strain carrying mutation rdn15 exhibits extremely high sensitivity toward paromomycin and also increases sensitivity of yeast ribosomes to tobramycin but to a lesser degree. These results demonstrate the ability of this mutation to increase affinity of the A-site for aminoglycosides. In contrast, mutation rdn15 causes resistance to hygromycin, revealing that binding and possibly action of hygromycin is differentiated from the other two aminoglycosides. The strain carrying mutation rdnhyg1 is resistant to all three antibiotics tested compared to wild type, indicating that U1495 participates in aminoglycoside binding to the ribosome. Cells carrying the extraribosomal mutation sup45-R2ts are more sensitive toward all three antibiotics compared to their wild type cells. Nevertheless, mutation sup45-R2ts does not affect the ability of these antibiotics to bind to the ribosome and directly induce translational errors. The study of amingolycoside action confirmed that paromomycin and hygromycin inhibit cells growth, while no such effect is observed during cell growth in the presence of tobramycin. This fact is combined with the inability of tobramycin to inhibit substrate binding to the ribosomal A-site Nevertheless, it was shown that tobramycin, like paromomycin and hygromycin, is effective both in inducing translational errors and increasing polyphenylalanine synthesis in wild-type and mutant ribosomes.

Ο σκοπός της παρούσας διατριβής εστιάζεται στη διερεύνηση της λειτουργίας και της δομής του ευκαρυωτικού ριβοσώματος. Συγκεκριμένα, ερευνά την επίδραση ορισμένων συστατικών του ριβοσωματικού RNA (rRNA) της μεγάλης και της μικρής υπομονάδας του ριβοσώματος αλλά και ορισμένων εξωριβοσωματικών συστατικών επί της πιστής αποκωδικοποίησης της γενετικής πληροφορίας, επί της ικανότητας κατάλυσης του σχηματισμού πεπτιδικού δεσμού, αλλά και επί της μετατόπισης του πεπτιδυλο-tRNA από την Α στην Ρ ριβοσωματική περιοχή. Τέλος, διερευνήθηκε για πρώτη φορά σε ευκαρυωτικά κύτταρα, η πιθανότητα συσχέτισης της πιστότητας με την οποία επιτελούν τα κύτταρα τη μετάφραση με την οξειδωτική τους κατάσταση. Η μεθοδολογία που αναπτύχθηκε περιλαμβάνει α) τον προσδιορισμό της συχνότητας λάθους (E.F) in vitro, η οποία αποτελεί μέτρο της πιστότητας της μετάφρασης, β) τον προσδιορισμό της ταχύτητας κατάλυσης του σχηματισμού πεπτιδικού δεσμού που αποτελεί μέτρο της ενεργότητας της ριβοσωματικής πεπτιδυλοτρανσφεράσης και γ) την εξάρτηση του σταδίου μετατόπισης από την συγκέντρωση των διαλυτών πρωτεϊνικών παραγόντων και του κυκλοεξιμιδίου. Επιπλέον, διερευνήθηκε η επίδραση ορισμένων αντιβιοτικών, κυρίως της παρομομυκίνης και του κυκλοεξιμιδίου, in vivo και in vitro. Επίσης, προσδιορίστηκε η ικανότητα συγκρότησης των ριβοσωματικών υπομονάδων, των ακέραιων ριβοσωμάτων και των πολυσωμάτων. Τέλος, προσδιορίστηκαν χαρακτηριστικοί δείκτες της οξειδωτικής κατάστασης του κυττάρου. Παρά το γεγονός ότι συστατικά του rRNA είναι κυρίως υπεύθυνα για τη ριβοσωματική λειτουργία, τα αποτελέσματα της πρώτης ενότητας οδηγούν στο συμπέρασμα ότι η πιστότητα της μετάφρασης, η καταλυτική ενεργότητα αλλά και το στάδιο μετατόπισης της μετάφρασης καθορίζονται ως ένα βαθμό και από εξωριβοσωματικά συστατικά όπως είναι η φωσφατάση σερίνης/θρεονίνης SAL6 και το προϊόν του γονιδίου ASU9. Τα συμπεράσματα της δεύτερης θεματικής ενότητας δίνουν έμφαση στην συνεχή ενδοεπικοινωνία που υφίσταται μεταξύ των δυο ριβοσωματικών υπομονάδων. Πράγματι, ορισμένες μεταλλάξεις στο rRNA της μικρής υπομονάδας του ριβοσώματος επηρεάζουν εκτός από την κύρια λειτουργία αυτής, δηλαδή την αποκωδικοποίηση, και την ταχύτητα σχηματισμού πεπτιδικών δεσμών. Αντίστροφα, ορισμένες μεταλλάξεις στο rRNA της μεγάλης υπομονάδας του ριβοσώματος επηρεάζουν εκτός από την κύρια ενεργότητα αυτής, δηλαδή την ενεργότητα πεπτιδυλοτρανσφεράσης, και την πιστότητα της αποκωδικοποίησης. Ορισμένες εξ αυτών των μεταλλάξεων επηρεάζουν επίσης και το στάδιο της μετατόπισης. Στην τρίτη ενότητα αποδεικνύουμε ότι οι επιρρεπείς σε λάθη μεταλλάξεις παρουσιάζουν χαμηλότερο οξειδωτικό στρες ενώ οι υπερακριβείς μεταλλάξεις παρουσιάζουν υψηλότερο οξειδωτικό στρες. Μια ελκυστική ερμηνεία των αποτελεσμάτων είναι ότι όσο πιο υπερακριβές είναι ένα κύτταρο κατά τη μετάφραση τόσο περισσότερο είναι το ποσό της ενέργειας που πρέπει να καταναλώσει ώστε να εξασφαλίσει την αποφυγή λαθών κατά τη πρωτεϊνοσύνθεση, ελαττώνοντας κατά αυτό τον τρόπο το ποσό ενέργειας με το οποίο θα αντιμετωπίζονταν οι ελεύθερες ρίζες. / The aim of the present diatribe focuses on the function and the structure of the eukaryotic ribosome. Specifically, the influence of several ribosomal RNA (rRNA) residues from the small and the large ribosomal subunit as well as the influence of several extra-ribosomal elements on the accurate decoding of the genetic information, on the catalysis of peptide bond formation and on the translocation of peptidyl-tRNA from the A to the P ribosomal site, is investigated. In the last part, the possible correlation between translational fidelity and the cell’s oxidative status is determined for the first time in eukaryotic cells. The methodology that was applied includes a) the error frequency (E.F) determination that measures translational fidelity, b) the determination of the catalytic rate constant for peptide bond formation that reflects the ribosomal peptidyltransferase activity and c) the dependence of the translocation step on soluble protein factors concentration and on cycloheximide concentration. Moreover, we studied the effects of the antibiotics paromomycin and cycloheximide in vivo and in vitro. The assembly of ribosomal subunits, of ribosomes and of polysomes was also investigated. Finally, typical markers of the cell’s oxidative status were determined. Despite the fact that rRNA residues are mainly responsible for ribosomal function, the results from the first part of the thesis lead to the conclusion that the translational fidelity, the catalytic activity and the translocation step of translation are determined up to a certain level by extra-ribosomal elements such as the serine/threonine phosphatase SAL6 as well as the ASU9 gene product. The conclusions drawn from the second part of the diatribe point to the constant intercommunication between the two ribosomal subunits. Indeed, several mutations in the small ribosomal subunit rRNA not only affect its major function, i.e. the decoding process, but they also affect the rate of peptide bond formation. Reversely, several mutations in the large ribosomal subunit rRNA not only affect its major activity, i.e. the peptidyltransferase activity, but they also affect the accuracy of decoding. Some of these mutations influence also the translocation step of protein synthesis. In the third part, we prove that error-prone mutations display lower oxidative stress whereas the hyperaccurate mutations display higher oxidative stress. An attractive interpretation of these results is that a cell might spend more energy in order to achieve hyperaccuracy during translation thus reducing the amount of energy left in order to combat free radicals.