Pseudouridine synthases are enzymes responsible for modifying uridines to pseudouridines in a site-specific and energy-independent manner. There are 5 families of these synthases, named after the first member of each family to be characterized in Escherichia coli : RluA, RsuA, TruA, TruB and TruD. The 23S ribosomal RNA in E. coli contains 10 pseudouridine modifications made by 6 specific synthases named RluA-RluF. These modifications cluster around important functional regions of the ribosome such as the peptidyl transferase center, the tRNA binding sites, and the inter-subunit bridge regions. My research focuses on understanding the mechanisms of substrate selection by pseudouridine synthases and the roles of these modifications in ribosome biogenesis and function. The main aims of my research were: a) to examine the substrate specificity determinants of RluD, an important E. coli synthase; b) to characterize a mutant strain of E. coli lacking a majority of the pseudouridines on 23S rRNA; and c) to determine the activity of RluA from Vibrio cholerae, which has two closely related RluA paralogs rather than just one, as seen in most organisms. Pseudouridine modifications in the stem-loop of helix 69 (H69) in domain IV of 23S ribosomal RNA are highly conserved in all phyla. The three pseudouridines in H69 in E. coli have been shown to play an important role in 50S subunit assembly and its association with the 30S subunit. These three modifications are made by the pseudouridine synthase, RluD. Previous work showed that RluD is required for normal ribosomal assembly and function, and is the only pseudouridine synthase required for normal growth in E. coli. Here, we show that RluD is far more efficient in modifying H69 in structured 50S subunits, rather than in naked or synthetic 23S rRNA. We suggest that pseudouridine modifications in H69 are made late in the assembly of 23 rRNA into the mature 50S subunit. This is the first reported observation of a pseudouridine synthase being able to modify a structured ribonucleoprotein particle, and may constitute an important late step in the maturation of 50S ribosomal subunits. Deletion of RluD results in aberrant ribosome assembly and impaired translation termination leading to severe growth defects. However, single deletion strains of the remaining five 23S rRNA synthases do not display an altered phenotype. In an effort to identify possible roles for the remaining seven pseudouridines, we constructed a strain (Delta 5 mutant) lacking all 23S rRNA synthases except RluD. Surprisingly, this strain does not exhibit a significant growth defect at 37C in rich or minimal media. However, it does display a slower growth rate at 20C compared to wild-type. When grown in competition with the wild-type strain at 37C, a strong selection against the mutant strain was observed. In order to evaluate the structure of the mutant ribosomes, we determined the effect of various antibiotics that target the 50S subunit. The mutant strain is significantly more sensitive than wild-type to antibiotics targeting the 50S subunit such as chloramphenicol, hygromycin, clindamycin and tiamulin but these effects can be attributed to the loss of the RluC modification at U2504 by itself. In phenotypic microarray tests, we observed that the Delta 5 mutant grew much poorer than wild-type when cultured in a medium containing 6% NaCl. Taken together, the data suggest that these pseudouridines may play an important role in maintaining the structural integrity of the ribosome. In E. coli, the pseudouridine synthase RluA is a dual specificity synthase capable of modifying U746 on 23S rRNA and U32 on 4 cytoplasmic tRNAs. Surprisingly, the Vibrio cholerae genome encodes not one, but two closely related RluA proteins. In order to examine the possible activities of these two proteins, we complemented an rluA deletion in E. coli with plasmid-borne Vibrio rluA1 and rluA2 constructs. Interestingly, only one of these two RluA proteins (Vibrio RluA1) was able to modify E. coli 23S rRNA at U746. In order to determine the structural basis for this difference between the closely related RluA1 and RluA2, we constructed homology models using the structure of E. coli RluA in complex with an RNA stem-loop (PDB ID: 2I82) as a template. These models implicated two possible three amino acid (GVF or FAL) inserts present near the catalytic aspartate in Vibrio RluA2 as the likely cause of the differential activity. We hypothesize that this insert may sterically occlude the binding of substrate RNA to the enzyme, thereby preventing a productive modification reaction.

La réponse stringente est un processus mis en place lors d'une carence nutritionnelle qui permet l'arrêt coordonné de la croissance. Cette réponse essentielle à la survie des bactéries est très conservée. Elle se caractérise par la production et l'accumulation de guanosine tretra- et pentaphosphate (ppGpp). Le ppGpp, en se fixant sur l'ARN polymérase modifie ses propriétés cinétiques et affecte ainsi de manière globale la transcription de très nombreux gènes. Principalement, l'accumulation de ppGpp inhibe la biosynthèse des ARNs stables (ARNr et ARNt) et en conséquence inhibe la biogenèse des ribosomes. Chez Escherichia coli, le niveau de ppGpp est régulé par les deux enzymes RelA et SpoT. Lors d'une carence en acides aminés, RelA fixée au ribosome détecte le blocage de la machinerie traductionnelle causée par la fixation d'un ARNt déacylé au site A du ribosome, et synthétise du ppGpp. SpoT quant à elle serait capable de détecter et de synthétiser le ppGpp en réponse à d'autres carences nutritionnelles notamment en source de carbone, mais les mécanismes et les signaux détectés sont inconnus. Il a été proposé que la protéine CgtA serait impliquée dans le contrôle de la réponse stringente, en interagissant avec SpoT au niveau des ribosomes. CgtA est une GTPase conservée et essentielle de la famille Obg, mais sa fonction précise est inconnue. Elle a été impliquée à la fois dans la maturation des ribosomes et dans la ségrégation des chromosomes et la division. Le gène cgtA est situé en aval des gènes rplU, rpmA, et yhbE codant respectivement pour les protéines L21 et L27 de la sous-unité 50S du ribosome et pour une protéine intégrale de membrane interne de fonction inconnue. / The stringent response is a physiological process that occurs when bacterial cells encounter nutritional stresses, and allowing coordinated growth arrest. This conserved response is characterized by the accumulation of tetra- and pentaphosphate guanosine (ppGpp). ppGpp bind to RNA polymerase and modifies its kinetic properties, thereby affecting the transcription of many genes. Prinicpaly, ppGpp accumulation inhibits stable RNAs (rRNA and tRNA) biosynthesis, which in consequence inhibits ribosome biogenesis. Escherichia coli contains two enzymes involved in ppGpp metabolism, RelA and SpoT. During amino acid starvation, RelA bound to ribosomes produces ppGpp in response to the presence of uncharged tRNA in the ribosomal A-site. In contrast, SpoT produces ppGpp in response to other types of nutrient limitations, such as carbon starvation, but the detected signals and mechanism involved are still unknown. It has been proposed that the CgtA protein is involved in the stringent response control by interacting with SpoT at the ribosome. CgtA is a conserved and essential small GTPase of the Obg family. CgtA has also been implicated in ribosome maturation, chromosome segregation and division, but its precise function remains unknown. The cgtA gene is located downstream of rplU, rpmA and yhbE genes coding respectively for L21 and L27 proteins of the 50S subunit of the ribosome, and an integral inner membrane protein of unknown function. This genetic proximity with rplU and rpmA genes is highly conserved in bacteria. My thesis work was therefore organized around three questions. First, understanding the role of CgtA in growth control and in the stringent response.