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Ana Rita Macedo Bezerra Genómica Molecular De Uma Alteração Ao

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Ana Rita Macedo Bezerra Genómica Molecular De Uma Alteração Ao Universidade de Aveiro Departamento de Biologia 2013 Ana Rita Macedo Genómica molecular de uma alteração ao código Bezerra genético. Molecular genomics of a genetic code alteration. Universidade de Aveiro Departamento de Biologia 2013 Ana Rita Macedo Genómica molecular de uma alteração ao código Bezerra genético. Molecular genomics of a genetic code alteration. Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Biologia, realizada sob a orientação científica do Prof. Doutor Manuel António da Silva Santos, Professor Associado do Departamento de Biologia da Universidade de Aveiro Apoio financeiro da FCT e do FSE no âmbito do III Quadro Comunitário de Apoio. o júri presidente Doutora Maria Hermínia Deulonder Correia Amado Laurel Professora Catedrática da Universidade de Aveiro Doutora Judith Berman Professora Catedrática da Universidade de Tel Aviv Doutora Margarida Paula Pedra Amorim Casal Professora Catedrática da Universidade do Minho Doutor Manuel António da Silva Santos Professor Associado da Universidade de Aveiro Doutora Isabel Antunes Mendes Gordo Investigadora Principal do Instituto Gulbenkian de Ciência Doutor António Carlos Matias Correia Professor Catedrático da Universidade de Aveiro agradecimentos First and foremost, I would like to thank my supervisor, Doutor Manuel Santos, for the opportunity to work on this project and for his support throughout the last 5 years. Thank you for keeping me going when times were tough, asking acknowledgements insightful questions, and offering invaluable advice whilst allowing me the room to work in my own way. I am indebted to many colleagues who helped me during these last 5 years, especially to João Simões whose precious input was essential for this work. To Tatiana, Cristina, Denisa and João Paredes for helping me when I started my laboratory work. To Ana, Marisa, Tobias, Laura, Céu and Patrícia for support and fruitful discussions during my journey in the laboratory. To Violeta for the constant patience and readiness with everything and to the other past members of the Aveiro RNA Biology laboratory for their friendship and for making the laboratory such a pleasant place to do science. My gratitude goes also to my international colleagues of the EU-FP7 Sybaris consortium that helped with the Candida albicans whole genome sequencing and data analysis. In particular, Wanseon Lee and Johan Rung from the European Bioinformatics Institute (EBI-EMBL) in Cambridge, Ivo Gut and Mónica Bayés from Centro Nacional de Análises Genómico (CNAG) in Barcelona and Lisa Rizzetto from Florence University in Florence. And also to Professor Judith Berman from the Universities of Telavive and Minnesota for the constructive comments and discussions about my work. I am grateful to FCT, for funding my work through a PhD grant SFRH/BD/39030/2007 and project PTDC/SAU-GMG/098850/2008. Finally, I am deeply grateful to my parents, who stood by me and supported my choices and to my sister for her encouragement and care. A very special thanks to all my friends, who were always a phone call away, in particular to Renato for all the listening and advising. A big thank you to all of you. palavras-chave Candida albicans, código genético, tRNA, evolução, erros de tradução, transcriptoma, genoma resumo O código genético não é universal. Alterações à identidade de vários codões descobertas em procariotas e eucariotas invalidam a hipótese dum código genético universal e imutável. Por exemplo, várias espécies do género Candida traduzem o codão CUG de leucina como serina. Em Candida Ser albicans, um único tRNA de serina (tRNACAG ) incorpora in vivo 97% de serina e 3% de leucina nas proteínas em resposta a codões CUG presentes nos mRNAs deste fungo patogénico. Esta ambiguidade é flexível e a incorporação de leucina aumenta em condições de stress. De forma a elucidar a função desta ambiguidade e determinar se a identidade dos codões CUG podia ser revertida de serina para leucina, desenvolvemos uma estratégia de evolução forçada e uma proteína recombinante fluorescente cuja actividade depende da incorporação de leucina num codão CUG. Construímos estirpes que incorporam leucina nas proteínas em resposta a codões CUGs em níveis que variam entre 0,64% e 98,46%. Esta reversão de uma alteração ao código genético demostrou de modo inequívoco que o código é flexível e pode evoluir. Testes de crescimento em diferentes meios de cultivo revelaram uma série impressionante de fenótipos com elevado potencial adaptativo nas estirpes mais ambíguas, nomeadamente tolerância a antifúngicos. A sequenciação dos genomas das estirpes que construímos revelou que a ambiguidade do codão CUG resulta na acumulação de polimorfismos de nucleótido únicos (SNP) no genoma. Verificámos também perda de heterozigozidade (LOH) nos cromossomas 5 e R das estirpes que incorporam 80,84% e 98,46% de leucina em locais proteicos codificados por codões CUG. Os SNPs acumularam-se preferencialmente em genes envolvidos na adesão celular, no crescimento filamentoso e na formação de biofilmes, sugerindo que C. albicans utiliza a sua ambiguidade natural para aumentar a diversidade genética dos processos relacionados com a patogénese e resistência a drogas. Estes resultados evidenciam uma notável flexibilidade do código genético de C. albicans e revelam funções inesperadas da ambiguidade do código genético na evolução da diversidade genética e fenotípica. keywords Candida albicans, genetic code, tRNA, evolution, mistranslation, transcriptome, genome abstract The genetic code is not universal. Alterations to its standard form have been discovered in both prokaryotes and eukaryotes and demolished the dogma of an immutable code. For instance, several Candida species translate the standard leucine CUG codon as serine. In the case of the human pathogen Ser Candida albicans, a serine tRNA (tRNACAG ) incorporates in vivo 97% of serine and 3% of leucine in proteins at CUG sites. Such ambiguity is flexible and the level of leucine incorporation increases significantly in response to environmental stress. To elucidate the function of such ambiguity and clarify whether the identity of the CUG codon could be reverted from serine back to leucine, we have developed a forced evolution strategy to increase leucine incorporation at CUGs and a fluorescent reporter system to monitor such incorporation in vivo. Leucine misincorporation increased from 3% up to nearly 100%, reverting CUG identity from serine back to leucine. Growth assays showed that increasing leucine incorporation produced impressive arrays of phenotypes of high adaptive potential. In particular, strains with high levels of leucine misincorporation exhibited novel phenotypes and high level of tolerance to antifungals. Whole genome re-sequencing revealed that increasing levels of leucine incorporation were associated with accumulation of single nucleotide polymorphisms (SNPs) and loss of heterozygozity (LOH) in the higher misincorporating strains. SNPs accumulated preferentially in genes involved in cell adhesion, filamentous growth and biofilm formation, indicating that C. albicans uses its natural CUG ambiguity to increase genetic diversity in pathogenesis and drug resistance related processes. The overall data provided evidence for unantecipated flexibility of the C. albicans genetic code and highlighted new roles of codon ambiguity on the evolution of genetic and phenotypic diversity. Contents List of contents List of figures…………………………………………………………………………... ii List of tables……………………………………………………………………………. ii List of abbreviations………………………………………………………………….. iv Chapter 1 Introduction……………………………………………………………………………... 1 1.1 Genetic code: the interface between nucleic acid and protein chemistry…….. 2 1.1.1 The standard genetic code………………………………………………….. 2 1.1.2 Genetic code components: AARS and tRNAs……………………………. 4 1.1.2.1 Molecular architecture of AARS…………………………………… 4 1.1.2.2 Structure and function of the tRNA molecule…………………….. 7 1.1.2.3 Aminoacylation reaction……………………………………………. 11 1.1.2.4 AARS:tRNA interactions……………………………………………. 12 1.1.2.5 Quality control in tRNA charging…………………………………... 15 1.2 Protein synthesis: the process of mRNA translation……………………………. 18 1.2.1 Mechanism of mRNA translation…………………………………………… 18 1.2.1.1 mRNA translation initiation…………………………………………. 19 1.2.1.2 mRNA translation elongation………………………………………. 21 1.2.1.3 mRNA translation termination and recycling……………………... 22 1.2.2 Ribosomal proofreading……………………………………………………... 24 1.2.3 mRNA mistranslation………………………………………………………… 26 1.3 Development of the genetic code…………………………………………………. 28 1.3.1 Origin and early evolution of the genetic code……………………………. 28 1.3.2 Natural variations in the universal assignment of codons……………….. 30 1.3.3 Natural expansion of the genetic code to 22 amino acids……………….. 32 1.3.4 Theories for the evolution of the genetic code……………………………. 35 1.3.5 Variation in codon assignment through codon identity engineering……. 38 1.4 Evolution of the genetic code in yeast……………………………………………. 40 1.4.1 Reassignment of the CUN codon family in yeast mitochondria………… 41 1.4.2 Reassignment of the UGA stop codon in yeast mitochondria…………... 41 1.4.3 Reassignment of the CUG codon in the CTG clade……………………… 42 Ser 1.4.3.1 tRNA CAG, LeuRS and SerRS…………………………………….. 43 1.4.3.2 Pathway for the CUG reassignment in the fungal CTG clade….. 45 1.4.4 C. albicans as a model system to study the genetic code………………. 47 1.4.4.1 C. albicans biology………………………………………………….. 49 1.5 Objectives of this study…………………………………………………………….
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