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The mRNA coding for the ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella was studied in induced and uninduced cells to determine the molecular mechanisms which regulate the cellular levels of this enzyme. A procedure for isolation of a high yield of total undegraded cellular polysomes was developed. The crosslinking reagent, dimethyl suberimidate, was employed to prepare a stable NADP-GDH-crosslinked-Sepharose-4B antigen affinity column for the purification of rabbit anti-NADP-GDH IgG. Binding studies with ¹²⁵I-labelled antibody and total polysomes, isolated from induced and uninduced cells, showed that the NADP-GDH was being synthesized on polysomes from both types of cells. When poly(A)- containing RNA was extracted from polysomes isolated from induced and uninduced cells, and translated in an mRNA-dependent in vitro translation system, NADP-GDH antigen was synthesized from the RNA from both sources. Based on sucrose density gradient analysis, Chlorella NADP-GDH mRNA has a sedimentation coefficient of 18 Comparison of the amounts of NADP-GDH synthesized in vitro from poly(A)-containing RNA and non-poly(A)-containing RNA showed the NADP-GDH mRNA contained polyadenylic acid sequence. By use of an indirect immunoadsorption procedure, the NADP-GDH mRNA was purified five- to sevenfold from total poly(A)-containing RNA. The overall purification of the NADP-GDH mRNA from total polysomal RNA was approximately two hundred-fold. Complementary DNA was synthesized from the partially purified RNA with reverse transcriptase. The cDNA sequences hybridized to the least abundant class of mRNA sequences present in total poly(A)-containing RNA. In vitro translation of total poly(A)-containing RNA showed that NADP-GDH synthesis was 0.1% of total protein synthesis. Upon addition of inducer to previously uninduced, synchronous cells, the amount of translatable NADP-GDH mRNA increased in a linear fashion after 30 min of the induction period. A change in rate of NADP-GDH mRNA accumulation was observed after 30 min of the induction period. The results support the prediction that since the NADP-GDH enzyme is unstable in vivo, during periods of NADP-GDH accumulation, the NADP-GDH mRNA accumulates. When poly(A)-containing RNA, isolated from uninduced synchronous cells was translated in vitro, NADP-GDH antigen was synthesized at each time in the cell cycle examined. The amount of translatable NADP-GDH mRNA increased throughout the cell cycle with a rate change occuring during the S-phase. This pattern of NADP-GDH mRNA accumulation is consistent with the hypothesis that NADP-GDH mRNA accumulates in uninduced cells at a rate proportional to gene dosage. These results provide one explanation for the observed pattern of enzyme potential in synchronous cells cultured in the absence of inducer. The data are consistent with the possibility that a single mRNA, which is subject to post-transcriptional modification by the inducer, codes for NADP-GDH. / Ph. D.

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.

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During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.<p>In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.<p>In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is distantly related to its yeast homologues which are 1-methylguanosine (m1G) specific. To understand the difference of activity between the Trm10 enzymes, we compared the yeast and the S. acidocaldarius Trm10 structures. Remarkably several Trm10 proteins (such as Trm10 of Thermococcus kodakaraensis) are even able to form both m1A and m1G. To understand the capacity of the T. kodakaraensis protein to methylate A and G, a mutational study was initiated./Lors de la traduction, les ARN de transfert (ARNt) jouent le rôle crucial d’adaptateurs entre l’ARN messager et les acides aminés. Les ARNt sont transcrits sous forme de pré-ARNt qui doivent être maturés. Lors de cette maturation, plusieurs nucléosides sont modifiés. Un grand nombre de ces modifications sont des méthylations des bases ou du ribose. Quatre familles d’ARNt méthyltransferases sont actuellement connues, dont la superfamille des SPOUT. Les membres de cette superfamille sont caractérisés par un nœud dans la chaîne polypeptidique du côté C-terminal. C’est au niveau de ce nœud que se lie la S-adénosylméthionine qui est le donneur de groupement méthyle. A l’exception de Trm10 qui est monomérique, toutes les protéines SPOUT connues sont dimériques et leur site actif est formé de résidus provenant des deux protomères. Selon l’espèce, une même modification peut être formée à la même position dans la molécule d’ARNt par des enzymes qui appartiennent à des familles différentes. A l’opposé, des enzymes homologues peuvent présenter des spécificités ou des activités différentes.<p>Au cours de ce travail, nous avons identifié l’enzyme TrmJ de Sulfolobus acidocaldarius (l’organisme modèle des Crénarchées hyperthermophiles) qui méthyle le ribose du nucléoside en position 32 des ARNt. Cette protéine est un homologue de l’enzyme TrmJ de la bactérie Escherichia coli. L’étude comparative que nous avons réalisée a révélé que ces deux enzymes présentent une différence de spécificité pour la nature du nucléoside en position 32 ainsi que pour les ARNt substrats. Afin de comprendre ces différences de spécificité au niveau moléculaire, les structures des domaines SPOUT des deux TrmJ ont été déterminées et comparées.<p>En parallèle, nous avons résolu la structure cristalline de la protéine Trm10 de S. acidocaldarius. C’est la première structure disponible d’un enzyme Trm10 formant de la 1-méthyladénosine (m1A). C’est aussi la première structure complète d’une protéine Trm10. Les enzymes homologues des levures Saccharomyces cerevisiae et Schizosaccharomyces pombe qui n’ont que peu d’identité de séquence avec l’enzyme de S. acidocaldarius, forment de la 1-méthylguanosine (m1G). Dans le but de comprendre comment ces enzymes homologues peuvent présenter des activités différentes, leurs structures ont été comparées. De manière surprenante, certains homologues de Trm10 (comme l’enzyme de l’Euryarchée Thermococcus kodakaraensis) sont capables de former du m1A et du m1G. Afin de mieux comprendre comment ces protéines sont capables de méthyler deux types de bases, nous avons initié l’étude de l’enzyme Trm10 de T. kodakaraensis par mutagenèse dirigée.<p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The work discussed in this thesis deals with the significance of initiator tRNA gene copy number in Escherichia coli. A summary of the relevant literature discussing the process of protein synthesis, initiator tRNA selection and gene redundancy is presented in Chapter 1. Chapter 2 describes the ‘Materials and Methods’ used in the experimental work carried out in this thesis. The next three chapters address the significance of initiator tRNA gene copy number in E. coli at three levels; at the level of the molecule (Chapter 3), at the level of the cell (Chapter 4) and at the level of the population (Chapter 5). At the end of the thesis are appended three publications, which include two papers where I have contributed to work not discussed in this thesis and one review article. A brief summary of chapters 3 to 5 is provided below: (i) Chapter 3: Can E. coli remain viable without the 3 G-C base pairs in initiator tRNA? Initiator tRNAs are distinguished from elongator tRNAs by several features key among which are the three consecutive and near universally conserved G-C base pairs found in the anticodon stem of initiator tRNAs. These bases have long been believed to be essential for the functioning of a living cell, both from in vitro and in vivo analysis. In this study, using targeted mutagenesis and an in vivo genetics based approach, we have shown that the 3 G-C base pairs can be dispensed with in E. coli, and the cell can be sustained on unconventional initiator tRNAs lacking the intact 3 G-C base pairs. Our study uncovered the importance of considering the relative amounts of molecules in a living cell, and their role in maintaining the fidelity of protein synthesis. (ii) Chapter 4: Can elongator tRNAs initiate protein synthesis? There are two types of tRNAs; initiator tRNA, of which there is one representative in the cell, and elongator tRNAs of which there are several representatives. In this study, we have uncovered initiation of protein synthesis by elongator tRNAs by depleting the initiator tRNA content in the cell. This raises the possibility that competition between initiator and elongator tRNAs at the P site of the ribosome occurs routinely in the living cell, and provides a basis for initiation at several 'start' sites in the genome that may not be currently annotated as such. We speculate that such a phenomenon could be exploited by the cell to generate phenotypic diversity without compromising genomic integrity. (iii) Chapter 5: How many initiator tRNA genes does E. coli need? E. coli has four genes that encode initiator tRNA, these are the metZWV genes that occur at 63.5 min in the genome, and the metY gene that occurs at 71.5 min in the genome. Earlier studies indicated that the absence of metY had no apparent impact on cell growth. In view of the importance of initiator tRNA gene copy number in maintaining the rate and fidelity of protein synthesis, we examined the fitness of strains carrying different numbers of initiator tRNA genes by competing them against each other in both rich and limited nutrient environments. Our results indicate a link between caloric restriction and protein synthesis mediated by the initiator tRNA gene copy number.

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All cellular RNAs contain numerous chemically modified nucleosides, but the largest number and the greatest variety are found in transfer RNA (tRNA). These modifications are posttranscriptionally introduced by modification enzymes during the complex process of tRNA maturation. The function of these modified nucleosides is not well known, but it seems that when present in the anticodon region, they play a direct role in increasing translational efficiency and fidelity, while modifications outside the anticodon region would be involved in the maintenance of the structural integrity of tRNA. Among the naturally occurring nucleoside modifications, base and ribose methylations are by far the most frequently encountered. They are catalyzed by tRNA methyltransferases (MTases), using generally the S-adenosyl-L-methionine (AdoMet) as methyl donor. Most of the knowledge about tRNA MTases comes from studies on bacterial and eukaryal model organisms, and very few informations are available about tRNA methylation in Archaea, particularly for thermophilic and hyperthermophilic Archaea whose GC-rich tRNAs are difficult to sequence. Nevertheless, some works on tRNA hydrolyzates from thermophiles or hyperthermophiles highlighted the presence of numerous methylated nucleosides. Furthermore, it has been shown that the only sequenced tRNA from an hyperthermophilic Archaea, the initiator methionine tRNA (tRNAiMet) from the Sulfolobus acidocaldarius, contains ten modified nucleosides, nine of them bearing a methylation on the base, on the ribose or on both base and ribose.<p>Of special interest is the modified nucleoside found at position 9 of this tRNA. It is an adenosine derivative, but the exact nature of the modification is unknown. In the yeast S. cerevisiae, some tRNAs with a guanosine at this position are methylated by the MTase Trm10p to form m1G9 (126). Since Trm10p-related proteins are found in hyperthermophilic archaea, such a homolog could be responsible for modification at position 9 of S. acidocaldarius tRNAiMet. In this work, we showed indeed that the Trm10p-related protein Saci_1677p from S. acidocaldarius methylates position 9 of tRNAs, but is specific for position N1 of adenosine, forming m1A rather than m1G. Interestingly, we demonstrated that Tk0422p from T. kodakaraensis, the euryarchaeal homolog to Saci_1677p, is the first tRNA MTase presenting a broadened nucleoside recognition capability, methylating both position N1 of A and of G to form m1A and m1G at position 9 of tRNAs. <p>This unique tRNA (m1A-m1G) MTase activity was further studied on one hand by site-directed mutagenesis of residues potentially important for the catalytic activity of Tk0422p enzyme, and on the other hand by determining the importance of the pH on the efficacy of the methylation reaction. Indeed, protonation state of atom N1 of A and G differs at physiological pH (N1 of G being protonated contrary to N1 of A), and we showed that m1G formation was increased with increasing pH. This could reflect the need of the enzyme to deprotonate G to be able to catalyze de methyltransfer. We showed also that the activity of the two archaeal enzymes (Saci_1677p and Tk0422p) present different dependence toward the structure of tRNA, the euryarchaeal Tk0422p requiring the intact tRNA structure while its crenarchaeal counterpart Saci_1677p being able to modify some truncated tRNAs.<p>Finally, some attempts to unveil the in vivo function of these enzymes, as well as their enzymatic mechanisms were undertaken, but these experiments are very preliminary and underline the needs for the development of genetic tools applicable to Archaea./ Tous les ARN cellulaires contiennent des nucléosides modifiés chimiquement, mais ce sont les ARNt qui en contiennent la plus grande variété et la plus grande proportion. Ces modifications sont introduites post-transcriptionnellement par des enzymes de modification durant le processus complexe de maturation des ARNt. Parmi les nucléosides modifiés, les méthylations de bases ou de riboses sont les plus fréquemment rencontrées. Elles sont catalysées par des ARNt méthyltransférases (MTases) utilisant pour la plupart de la S-adenosyl-L-methionine (AdoMet) comme donneur de méthyle. <p>La plupart des connaissances relatives aux ARNt MTases provient d’études sur des organismes modèles eucaryotes et bactériens, et peu de choses sont connues en ce qui concerne les archées, plus particulièrement les archées thermophiles et hyperthermophiles dont les ARNt GC riches sont difficiles à séquencer. Néanmoins, des travaux sur des hydrolysats d’ARNt de thermophiles et hyperthermophiles ont mis en évidence la présence d’un grand nombre de nucléosides modifiés. De plus, le seul ARNt d’archée hyperthermophile séquencé à ce jour, l’ARNtiMet de S. acidocaldarius contient 10 nucléosides modifiés, essentiellement par méthylation de la base, du ribose, ou des deux à la fois. Le nucléoside présent en position 9 de cet ARNt porte une modification chimique de nature encore inconnue. Or, chez la levure S. cerevisiae, certains ARNt possédant une guanosine à cette position sont méthylés par la MTase Trm10p pour former la 1-méthylguanosine. Etant donné qu’il existe une protéine apparentée à Trm10p chez les archées hyperthermophiles, celle-ci pourrait être responsable de la modification trouvée en position 9 de l’ARNtiMet de S. acidocaldarius. Dans ce travail, nous avons montré qu’effectivement la protéine Saci_1677p de la crénarchée S. acidocaldarius, orthologue à Trm10p, modifie la position 9 des ARNt, mais catalyse la formation de 1-methyladénosine (m1A) plutôt que de m1G dans les ARNt. De façon intéressante, nous avons montré que chez l’euryarchée T. kodakaraensis, l’enzyme Tk0422p homologue à Saci_1677p est capable de méthyler à la fois une adénosine et une guanosine en position 9 des ARNt. A notre connaissance, cette enzyme est la première ARNt MTase présentant une capacité élargie de reconnaissance de substrat.<p>Le présent travail a contribué à la caractérisation fonctionnelle et structurale de ces deux enzymes archéennes, et a permis d’améliorer la connaissance générale de la machinerie de modification des ARNt d’archées.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished