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Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS. / We are studying the Neurospora VS ribozyme as a model system to increase our knowledge of the structure-function relationship in RNA and to better understand the mechanism of the cleavage reaction. It has been previously postulated that the A730 internal loop of stem-loop VI (SLVI) forms the active site of the VS ribozyme and binds magnesium ion(s) that may participate in catalysis. To get insights into the catalytic mechanism, we have determined by NMR spectroscopy the structure of a SLVI fragment containing the A730 loop. The structure we obtained is in agreement with previous biochemical studies and contains one or more magnesium-ion binding sites in the active site. Based on kinetic and mutagenesis studies, it has been proposed that an adenine in the A730 loop, A756, is important for catalysis and may participate in general acid/base catalysis. Previous pH-dependent enzymatic studies identified a catalytic pKa of 5.2-5.8, which likely corresponds to the protonation equilibrium of this A756 adenine in the A730 loop. Using 13C NMR methods, we have identified a shifted pKa for A756, which gives additional support to the role of this residue in the general acid/base mechanism. The NMR studies presented here therefore increase our understanding of the cleavage reaction in the VS ribozyme.

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LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.