Cuscuta pentagona is an obligate parasitic plant that hinders production of crops throughout the world. Parasitic plants have unique morphological and physiological features, the most prominent being the haustorium, a specialized organ that functions to connect them with their host's vascular system. The Cuscuta haustorium is remarkable in that it enables mRNA movement to occur between hosts and parasite, but little is known about the mechanisms regulating cross-species mRNA transfer or its biological significance to the parasite. These questions were addressed with genomics approaches that used high throughput sequencing to assess the presence of host mRNAs in the parasite as well as parasite mRNAs in the host. For the main experiment Cuscuta was grown on stems of Arabidopsis thaliana and tomato (Solanum lycopersicon) hosts because the completely sequenced genomes of these plants facilitates identification of host and parasite transcripts in mixed mRNA samples. Tissues sequenced included the Cuscuta stem alone, the region of Cuscuta-host attachment, and the host stem adjacent to the attachment site. The sequences generated from each tissue were mapped to host reference genes to distinguish host sequences, and the remaining sequences were used in a de novo assembly of a Cuscuta transcriptome. This analysis revealed that thousands of different Arabidopsis transcripts, representing nearly half of the expressed transcriptome of Arabidopsis, were represented in the attached Cuscuta. RNA movement was also found to be bidirectional, with a substantial proportion of expressed Cuscuta transcripts found in host tissue. The mechanism underlying the exchange remains unknown, as well as the function of mobile RNAs in either the parasite or host. An approach was developed to assay potential translation of host mRNAs by detecting them in the Cuscuta translatome as revealed by sequencing polysomal RNA and ribosome-protected RNA. This work highlights RNA trafficking as a potentially important new form of interaction between hosts and Cuscuta. / Ph. D.

Protein synthesis is a process by which organisms manufacture their proteins that perform various cellular activities either alone or in combination with other similar or different molecules. In eubacteria, protein synthesis proceeds at a rate of around 15 amino acids per second. The ribosomes, charged tRNAs and mRNAs can be considered as the core components of protein synthesis system which, in addition, involves a panel of non-ribosomal proteins that regulate the speed, specificity and accuracy of the process. Peptidyl-tRNA hydrolase (Pth) and ribosome recycling factor (RRF) are two such non-ribosomal proteins involved in protein synthesis. These two proteins are essential for eubacterial survival and the work reported in this thesis involves structural characterization of these two proteins from the bacterial pathogen, Mycobacterium tuberculosis. The protein structures were solved using established techniques of protein crystallography. Hanging drop vapour diffusion method and crystallization under oil using microbatch plates were the methods employed for protein crystallization. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator in all the cases. The data were processed using DENZO and MOSFLM. The structures were solved by molecular replacement method using the program PHASER. Structure refinements were carried out using programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, CHIMERA, and PYMOL were used for structure validation and analysis of the refined structures. Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA that has dropped off from ribosome before reaching the stop codon, in order to avoid the toxicity resulting from peptidyl-tRNA accumulation and to free the tRNA to make it available for further rounds in protein synthesis. To begin with, the structure of the enzyme from M. tuberculosis (MtPth) was determined in three crystal forms. This structure and the structure of the same enzyme from Escherichia coli (EcPth) in its crystal differ substantially on account of the binding of the C-terminus of the E.coli enzyme to the peptide binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in open conformation when the enzyme is in the free state as in the crystals of MtPth. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the EcPth crystal mimics the peptide-bound conformation of the enzyme. The peptide stretch involving a loop and a helix, referred to earlier, now closes on the bound peptide. Concurrently, a gate connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of the two residues. Thus, the crystal structure of MtPth when compared with that of EcPth, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. A discrepancy between the X-ray results and NMR results, which subsequently became available, led to X-ray studies on new crystal forms of the enzyme. The results of these studies and those of the enzyme from different sources that became available, confirmed the connection deduced previously between the closure of the lid at the peptide-binding site and the opening of the gate that separates the peptide-binding site and tRNA binding site. The plasticity of the molecule indicated by X-ray structures is in general agreement with that deduced from the available solution NMR results. The correlation between the lid and the gate movement is not, however, observed in the NMR structure of MtPth. The discrepancy between the X-ray and NMR structures of MtPth in relation to the functionally important plasticity of the molecule, referred to earlier, also led to molecular dynamics simulations. The X-ray and the NMR studies along with the simulations indicated an inverse correlation between crowding and molecular volume. A detailed comparison of proteins for which X-ray and the NMR structures are available appears to confirm this correlation. In consonance with the reported results of the investigation in cellular components and aqueous solutions, the comparison indicates that the crowding results in compaction of the molecule as well as change in its shape, which could specifically involve regions of the molecule important for function. Crowding could thus influence the action of proteins through modulation of the functionally important plasticity of the molecule. After termination of protein synthesis at the stop codon, the ribosome remains as a post-termination complex (PoTC), consisting of the 30S and the 50S subunits, mRNA and a deacylated tRNA. This complex has to be disassembled so that the ribosome is available for the next round of translation initiation. Ribosome recycling factor (RRF) binds to ribosome and in concert with elongation factor G (EF.G), performs the recycling of ribosome that results in disassembly of PoTC. The structure of this L-shaped protein with two domains connected by a hinge, from Mycobacterium tuberculosis (MtRRF) was solved previously in our laboratory. The relative movement of domains lies at the heart of RRF function. Three salt bridges were hypothesized to reduce the flexibility of MtRRF when compared to the protein from E.coli (EcRRF), which has only one such salt bridge. Out of these three bridges, two are between domain 1 and domain 2, whereas the third is between the hinge region and the C-terminus of the molecule. These salt bridges were disrupted with appropriate mutations and the structure and activity of the mutants and their ability to complement EcRRF were explored. An inactive C-terminal deletion mutant of MtRRF was also studied. Major, but different, structural changes were observed in the C-terminal deletion mutant and the mutant involving the hinge region. Unlike the wild type protein and the other mutants, the hinge mutant complements EcRRF. This appears to result from the increased mobility of the domains in the mutant, as evidenced by the results of librational analysis. In addition to the work on PTH and RRF, the author was involved during the period of studentship in carrying out X-ray studies of crystalline complexes involving amino acids and carboxylic acids, which is described in the Appendix of the thesis. The complexes studied are that of tartaric acid with arginine and lysine.

Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.

Το ριβόσωμα παρέχει την πλατφόρμα πάνω στη οποία το mRNA αναγνωρίζεται και αποκωδικοποιείται από τα μεταφορικά RNAs. Δρα καταλυτικά ως ριβοένζυμο και συνθέτει την αντίστοιχη αλληλουχία αμινοξέων. Σημαντικά λειτουργικό κομμάτι του ριβοσώματος αποτελεί το τούνελ εξόδου, το οποίο βρίσκεται στη μεγάλη υπομονάδα του ριβοσώματος και αποτελεί το κανάλι, μέσω του οποίου οι νεοσυντιθέμενες πεπτιδικές αλυσίδες εξέρχονται στο κυτταροδιάλυμα. Νεότερες ερμηνείες προσδίδουν στο τούνελ εξόδου έναν πιο ενεργό ρόλο, καταδεικνύοντας τη σπουδαιότητά του όχι μόνο ως διάδρομος εξόδου της πεπτιδικής αλυσίδας, αλλά και ως λειτουργική περιοχή του ριβοσώματος με σημαντικό ρόλο στη ρύθμιση της πρωτεϊνικής σύνθεσης. Το δίκτυο επικοινωνίας μεταξύ τούνελ και πεπτιδυλοτρανσφεράσης δεν έχει ακόμα χαρτογραφηθεί και μελετηθεί πλήρως, υπάρχουν όμως μέχρι στιγμής πολυπληθείς αναφορές που επιβεβαιώνουν ότι μέρος του αποτελούν κυρίως βάσεις του 23S rRNA, συστατικά του τοιχώματος της εισόδου στο τούνελ, και βάσεις ευρισκόμενες στο κέντρο της πεπτιδυλοτρανσφεράσης, συντηρημένες εξελεικτικά, γνωστές από την αλληλεπιδρασή τους με αναστολείς της πεπτιδυλοτρανσφεράσης και κυρίως με αναστολείς της οικογένειας των μακρολιδίων. Στην παρούσα διατριβή μελετήσαμε την αλληλεπίδραση των χαρακτηριστικών αυτών βάσεων που συγκροτούν το δίκτυο επικοινωνίας μεταξύ του τούνελ εξόδου και της πεπτιδυλοτρανσφεράσης με τη βοήθεια νέων μακρολιδίων τρίτης γενιάς, των κετολιδίων, που φέρουν επί πλέον και άτομα φθορίου, παρέχοντας έτσι στο μόριο μεγαλύτερη συγγένεια για φορτισμένες ομάδες. Σύμφωνα με τα αποτελέσματά μας, αποτελούν εξαιρετικά υποσχόμενους αντιμικροβιακούς θεραπευτικούς παράγοντες, και αναστέλλουν ισχυρά την μετάφραση. Παράλληλα καταλαμβάνουν αμοιβαία αποκλειόμενες θέσεις στο ριβόσωμα, στην είσοδο του τούνελ, αλληλεπιδρώντας με τις βάσεις Α2058, Α2059, Α2062 και Α752. Επειδή όλες οι παραπάνω βάσεις αποτελούν μέρος του δικτύου επικοινωνίας τούνελ-πεπτιδυλοτρανσφεράσης, θα μπορούσαμε να συμπληρώσουμε τον ήδη υπάρχοντα μηχανισμό δράσης των μακρολιδίων (drop-off, λόγω αύξησης της koff) με την επικοινωνία του τούνελ με την πεπτιδυλοτρανσφεράση, ώστε να απενεργοποιηθεί η τελευταία με τελική κατάληξη τη διακοπή της πρωτεϊνικής σύνθεσης και την επαγωγή του drop-off. Για την περαιτέρω μελέτη της αλληλεπίδρασης του τούνελ με την νεοσυντιθέμενη πεπτιδική αλυσίδα, σχεδιάσθηκαν νέα πεπτιδυλο παράγωγα της χλωραμφανικόλης, τα οποία προσομοιάζουν πεπτιδυλο-tRNAs προσδεδεμένα στην Α-θέση του ριβοσώματος, με την ολιγοπεπτιδική αλληλουχία να εκτείνεται βαθύτερα στο τούνελ, υιοθετώντας μία ανοικτή διαμόρφωση. Όπως διαπιστώσαμε με συναγωνιστικές μελέτες με ραδιενεργό ερυθρομυκίνη, τα συγκεκριμένα ανάλογα καταλαμβάνουν την Α-θέση στο κέντρο της πεπτιδυλοτρανσφεράσης, ενώ η συγγένεια του κάθε αναλόγου ως αναστολέα της λειτουργίας του ριβοσώματος, είναι απόλυτα ιδιοσυγκρατική, καθώς εξαρτάται από την αλληλουχία των αμινοξέων που συγκροτούν το πεπτίδιο. Μελετώντας το αποτύπωμα των ενώσεων αυτών στο ριβόσωμα, διαπιστώσαμε ότι ενώ η βάση της χλωραμφαινικόλης διατηρεί τη θέση της στη περιοχή Α, το πεπτίδιο εισέρχεται βαθειά στην είσοδο του τούνελ, αλληλεπιδρώντας με την βάση Α752. Συνεπώς τα πεπτιδυλο ανάλογα της χλωραμφαινικόλης, τα οποία συμπεριφέρονται ως ισχυροί αναστολείς της μετάφρασης, αποτελούν κατάλληλα εργαλεία μελέτης της αλληλεπίδρασης μεταξύ της νεοσυντιθέμενης πεπτιδικής αλληλουχίας και στοιχείων του τούνελ εξόδου του ριβοσώματος. / Ribosomes translate the genetic code into proteins in all living cells with extremely high efficiency, owing to their inherent flexibility and to their spectacular architecture. These large ribonucleoprotein particles synthesize proteins in all cells, using messenger RNA as the template, confirming that the ribosome is indeed a ribozyme, and aminoacyl-transfer RNAs as substrates. As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. New studies indicate that in some cases, the tunnel plays a more active role. Accumulated evidence had definitely concluded that ribosomal tunnel is not only a passive conduit for the nascent chains but a rather functionally important compartment, where specific peptide sequences establish direct interactions with the tunnel, talking back to the ribosome in order to regulate peptide synthesis, leading to translational stalling. In this study, we have investigated functional interactions between distinct locations of the ribosomal tunnel and specific residues of the peptidyltransferase, using new macrolides, known as ketolides, which carry also fluorine attached to the lactone ring either directly or indirectly. According to our results, the new antibiotics exhibited better antimicrobial activity, inhibiting strongly the translational apparatus and could be useful tools in the future for treatment of bacterial infections. Binding studies with radiolabeled erythromycin revealed that these drugs bound competitively with erythromycin at the large ribosomal subunit, with extremely low dissociation constants. In parallel, RNA footprinting indicated that the new ketolides occupy the main macrolide binding site in the ribosome, which is located at the entrance of the exit tunnel adjacent to the peptidyltransferase center. These drugs interact with A2058, A2059 and A2062, as well as their extended heteroaromatic alkyl-aryl side chain penetrates deeper in the tunnel protecting A752 of Helix 35, interacting with basepair A752-U2609. To explore further this interaction, we have synthesized new peptidyl conjugates of chloramphenicol, which resemble nascent peptidyl-tRNA chains bound to the A-site of the ribosome, with their peptide sequence located deeper within tunnel, adopting an extended configuration. According to our data, these compounds did indeed bind to the peptidyl transferase center, competing with radiolabelled- chloramphenicol for binding to the ribosome. The binding of each analog, as well as its inhibitory activity of ribosomal function, is absolutely idiosyncratic, depending on the sequence of amino acid of peptide moiety. Studying the footprinting pattern of these compounds on the ribosome, we confirmed that while the chloramphenicol base maintains its position on the A-site, the peptide moiety penetrates deeper in the entrance of the exit tunnel, interacting with nucleotide A752. Therefore, we believe that these chloramphenicol peptides could be useful tools for probing nascent polypeptide chain interaction with the ribosome.

No description available.

2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Paralogs are present during ribosome biogenesis as well as in mature ribosomes in form of ribosomal proteins, and are commonly believed to play redundant functions within the cell. Two previously identified paralogs are the protein pair Ssf1 and Ssf2 (94% homologous). Ssf2 is believed to replace Ssf1 in case of its absence from cells, and depletion of both proteins leads to severely impaired cell growth. Results reveal that, under normal conditions, the Ssf paralogs associate with similar sets of proteins but with varying stabilities. Moreover, disruption of their pre-rRNP particles using high stringency buffers revealed that at least three proteins, possibly Dbp9, Drs1 and Nog1, are strongly associated with each Ssf protein under these conditions, and most likely represent a distinct subcomplex. In this study, depletion phenotypes obtained upon altering Nop7, Ssf1 and/or Ssf2 protein levels revealed that the Ssf paralogs cannot fully compensate for the depletion of one another because they are both, independently, required along parallel pathways that are dependent on the levels of availability of specific ribosome biogenesis proteins. Finally, this work provides evidence that, in yeast, Nop7 is genetically linked with both Ssf proteins. / Les paralogues sont présents lors de la biogenèse des ribosomes ainsi que dans les ribosomes matures sous forme de protéines ribosomiques, et sont généralement censées jouer des fonctions redondantes dans la cellule. Deux paralogues précédemment identifiées sont la paire de protéines Ssf1 et Ssf2 (94 % d'homologie). Ssf2 remplacerait Ssf1 en cas d’absence du dernier dans la cellule, et l’absence des deux protéines diminue la croissance cellulaire. Nos résultats révèlent que, dans des conditions normales, les paralogues Ssf s’associent à des ensembles de protéines similaires, mais avec différentes stabilités. De plus, la perturbation de leurs particules pré-rRNP à l’aide de tampons de haute stringence a révélé qu'au moins trois protéines, probablement Dbp9, Drs1 et Nog1, sont fortement associées à chaque protéine Ssf dans ces conditions, et très probablement représentent des sous-complexes distincts. Dans cette étude, les phénotypes cellulaires observés lors de la déplétion des protéines Nop7, Ssf1 et/ou Ssf2 ont révélé que les paralogues Ssf ne peuvent pas compenser entièrement pour la diminution de l'autre, car ils sont, indépendamment l’un de l’autre, nécessaires le long de voies de biogénèse ribosomale parallèles qui dépendent des niveaux de protéines impliqués dans la biogénèse des ribosomes disponibles. Enfin, ce travail fournit des preuves que, dans la levure, Nop7 est génétiquement lié aux deux protéines Ssf.

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are post-translationally removed from L27 by a site-specific protease so that conserved residues important for tRNA stabilization at the peptidyl transferase center are exposed. We have identified a novel cysteine protease in S. aureus that performs this cleavage. This protease, which we have named Prp, is conserved in all bacteria containing the L27 N-terminal extension. L27 cleavage was shown to be essential in S. aureus; un-cleavable L27 did not complement an L27 deletion. Cleavage appears to play an essential regulatory role, as a variant of L27 lacking the cleavage motif could not complement. Ribosomal biology in eubacteria has largely been studied in E. coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of E. coli. This research lays the foundation for the development of new therapeutic approaches that target this novel, essential pathway.