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Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations. In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions. Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations. In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components. Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%.

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

This thesis describes experiments investigating the translational recoding activities and the novel dual signalling properties of eukaryotic ribosome skipping 2A sequences. Over twenty years ago, the 19 amino acid 2A region of a Picornavirus; namely, Foot-and-Mouth Disease Virus (FMDV) polypeptide was shown to possess apparent “self-cleaving” abilities, cutting at its own C-terminus during translation (Ryan et al., 1991). Active FMDV 2A-like sequences were subsequently found in a number of related viruses (Luke et al., 2008), with several now utilised as essential biotechnology multi-gene transfer tools (Luke et al., 2010b). Then, in 2006, eukaryotic 2A-like sequences were identified from trypanosome non-LTR sequences. These were found to be functional in vitro (Heras et al., 2006). I have been able to identify over 400 putative eukaryotic 2A-like sequences through searching the freely available online proteomic and genomic databases. Data is presented to show that these 2As were encoded in frame with non-LTRs, or metabolic, or immune function genes, from a wide range of eukaryotic organisms; but I could not discern any obvious phylogenetic distribution for 2A. I have discovered that the majority of eukaryotic 2A sequences tested can mediate ribosome skipping in vitro. Modelling in silico indicated that active 2A-like sequences possessed the propensity to form a central alpha-helical region, whereas the models suggested that inactive 2A-like sequences would be essentially unstructured. I also report that some of these eukaryotic 2A peptides constitute a novel form of dual protein targeting as they play a dual role as exocytic pathway signal peptides mediating extracellular protein trafficking. I have shown that this protein trafficking ability is evolutionarily conserved, with an echinoderm sequence able to direct protein targeting in both plant and mammalian cells. I therefore propose that these novel eukaryotic 2A sequences could potentially become extremely valuable in biotechnological engineering.

Antibiotic resistance threatens to throw mankind back into an era when infectious disease was the predominant cause of death. In an effort to mitigate this danger, we targeted ErmC’ and KsgA. Both methylate N6-adenosine of ribosomal RNA, though each serve different roles in their bacterial host. ErmC’ dimethylates A2058 on 23S rRNA, conferring resistance to MLSB antibiotics (macrolides, lincosamides, streptogramin B). KsgA aids in ribosome assembly, binding inactive 30S until dimethylating A1518/A1519 of 16S rRNA. Like most methyltransferases, ErmC’ and KsgA use cofactor S-adenosylmethionine (SAM) as their methyl source, which binds adjacent to their specific adenosine substrate. ErmC’ inhibitors could restore MLSB antibiotics against infections with this resistance mechanism. KsgA inhibitors could form novel antibiotics that stall 30S assembly. Previous studies reported a potent ErmC’ inhibitor, N6-cyclopentyl adenosine (1), binding to the substrate pocket with cyclopentyl bridging into the SAM pocket. We expanded this study by synthesizing 1 and 22 other N6-substituted analogs to explore more favorable interactions within the SAM pocket. When these compounds (1mM) were screened against ErmC’ and KsgA, some showed greater inhibition than 1. Two of these inhibitors that were crystallized with ErmC’, N6-8-octylamine adenosine (2.60Å) and N6-phenethyl adenosine (2.40Å), unexpectedly docked into the SAM pocket with their 5’-C pointing towards the substrate pocket. New compounds were made to exploit this orientation by adding substituents off the 5’-C to probe the substrate pocket. Through a five step synthesis, the 5’-OH of 1 was substituted with an amine linked to benzyl (30), phenethyl (31), propylphenyl (32) or butylphenyl (33). When 30-33 were screened using 20μM SAM, ErmC’ showed greater inhibition (relative to 1), while KsgA showed virtually none. However, when ErmC’ was tested using 0.5μM SAM, inhibition from 30-33 was nearly unchanged, whereas 1 became significantly more potent than 30-33, suggesting 30-33 were not binding to the SAM pocket. Preliminary data showed that raising 23S concentrations lowered inhibition from 32-33, while inhibition from 1, 30 and 31 was nearly unchanged, suggesting that at least 32-33 bound within the substrate pocket.

Members of the 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenase (2OG oxygenase) superfamily catalyse a wide range of oxidative reactions in biology. 2OG oxygenases require Fe(II) and atmospheric oxygen for their activity, and couple substrate oxidation with the decarboxylation of 2OG into succinate and carbon dioxide. There are more than sixty known 2OG oxygenases in the human genome; they modify small molecules, nucleic acids and proteins implicated in diverse biological processes. Importantly, the seemingly disparate functions of 2OG oxygenases often converge to regulate gene expression. 2OG oxygenases have been shown to affect epigenetic reprogramming, chromatin remodelling, transcription factor activity and mRNA splicing. Emerging evidence indicates that 2OG oxygenases are also involved in the translational control of gene expression. Oxygenases TYW5, ALKBH8, ALKBH5 and FTO were found to catalyse modifications of tRNA and mRNA. The work in this thesis extends these observations by demonstrating that 2OG oxygenase-catalysed protein hydroxylations also play an important role in protein synthesis. The catalytic activities of two oxygenases belonging to the JmjC-only family, NO66 and JMJD4, are described. NO66 catalyses the histidinyl hydroxylation of 60S ribosomal subunit protein L8. NO66 is part of a conserved group of ribosomal protein hydroxylases that can be traced back to prokaryotes. JMJD4 is a lysyl hydroxylase of eRF1, the eukaryotic release factor responsible for translation termination. The hydroxylation of eRF1 takes place on a conserved NIKS motif important for release factor activity, and promotes effcient translational termination. JMJD4 is further implicated in cell growth and cancer, though the link between its activity and tumourigenesis remains to be determined. These results highlight the potential of 2OG oxygenases as regulators of protein synthesis, and further extend the scope of 2OG oxygenase function. The small molecule inhibition of 2OG oxygenases presents a novel therapeutic possibility targeting translational control in cancer and other diseases.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.