This study examined the role of <I>PYKI </I>coding sequences in the expression of <I>PYK1::lacZ </I>gene fusions in <I>Saccharomyces cerevisiae. </I>Further aims were to examine the effects of the vector system upon the mRNA levels from these gene fusions the effect that these gene fusions have upon the yeast cell in general. Analysis of the <I>PYK1::lacZ </I>gene fusions revealed that <I>PYK1 </I>coding sequences were responsible for elevating mRNA levels. This elevation was not due to a single element within the coding region of the <I>PYK1 </I>gene as had been previously proposed (Purvis <I>et al.,</I> 1987a; Lithgow, 1989). Models for the stimulatory action of the <I>PYK1 </I>coding region upon the transcription of the <I>PYK1::lacZ</I> gene fusion were presented. <I>PYKI </I>coding region fragments in the <I>PYK1::lacZ</I> gene fusions stabilized the mRNA, but the data presented here were not consistent with a stability element within the <I>PYK1 </I>coding region. An alternative model was presented whereby the translation rate of the mRNA can influence its decay. The effect of expression of these gene fusions upon the yeast cell in general was monitored by examining the mRNA level of two chromosomal loci, <I>PYK1 </I>and <I>PGK1, </I>and by measuring the generation time. In contrast to previous findings, <I>PYK1 </I>and <I>PGK1 </I>mRNA levels were found not to change and so it was concluded that expression of these gene fusions had no general effect upon transcription or mRNA decay. However expression of these gene fusions did lead to an increase in generation time, and it was proposed that this might be due to a general effect upon translation brought about by a reduction in the intracellular pools of tRNAs for non-preferred codons.

Les complexes multi-protéiques jouent un rôle crucial dans les cellules vivantes en catalysant et servant d'intermédiaires entre pratiquement toutes les activités cellulaires essentielles. Cependant, un grand nombre de ces machines se trouvent en très faibles quantités dans les cellules en particulier en ce qui concernent les complexes eucaryotes. Ceci est réfractaire à leur extraction à grande échelle et empêche sévèrement l'élucidation de leur structure et fonction. Dans le but de rendre les complexes multi protéiques accessibles par la voie de production recombinante, le groupe Berger a mis au point un ensemble de systèmes d'expression sur mesure pour la surproduction de complexes multi protéiques dans différents organismes hôtes incluant E. coli, les cellules d'insectes et les cellules de mammifères. Ces systèmes et en particulier le système MultiBac baculovirus/cellules d'insecte ont d'ors et déjà grandement contribués à l'étude de l'assemblage structural et fonctionnel à l'échelle moléculaire et atomique de nombreux complexes multi protéiques importants. Cela inclut en particulier le facteur général humain de transcription TFIID, un complexe de ~1.5 MDa qui constitue le sujet de recherche du laboratoire Berger. Mes contributions dans le développement de la technologie pour la production et dans l'élucidation des complexes TFIID humains sont discutées en détails dans cette thèse. / Multiprotein complexes play a crucial role in living cells by catalyzing and mediating virtually all essential cellular activities. However, many of these essential machines exist in very low endogenous amount in cells, in particular for eukaryotic complexes. This is refractory to large-scale extraction from native source material, severely impeding the elucidation of their structure and function. In order to make multiprotein complexes accessible by means of recombinant production, the Berger laboratory has developed an array of advanced expression systems tailor-made for overproducing multiprotein complexes in various host organisms including E. coli, insect cells and mammalian cells. Those systems, in particular the MultiBac baculovirus/insect cell system have already greatly contributed to studying the structural and functional assemblies of numerous important multiprotein complexes in molecular and atomic detail. Notably, this includes also the human general transcription factor TFIID, a ~1.5 MDa complex, which is the research focus of the Berger laboratory. My contributions to the expression technology development and to the structural elucidation of human TFIID complexes are discussed in details in this thesis.

Trabalhos recentes indicam que a maior parte do transcriptoma de células de mamíferos é composto por RNAs não-codificadores de proteínas (ncRNAs). Nosso grupo tem identificado e caracterizado ncRNAs longos (>200 nt), sem splicing, expressos em regiões intrônicas de genes codificadores de proteína. Contudo, a biogênese, processamento e localização sub-celular desta classe de RNAs permanecem desconhecidos. Este trabalho teve como objetivos i) investigar a contribuição da RNA Polimerase II (RNAP II) na transcrição de ncRNAs intrônicos, ii) avaliar a meia-vida destes ncRNAs em relação a mRNAs, e iii) verificar a distribuição sub-celular de ncRNAs intrônicos. Os resultados obtidos indicaram que ncRNAs intrônicos são predominantemente transcritos pela RNAP II a partir de regiões promotoras funcionalmente semelhantes as que controlam a transcrição de mRNAs. Ensaios de estabilidade revelaram que, em média, ncRNAs intrônicos possuem meia-vida igual ou maior (3,4h a 4,2h) do que mRNAs (3,1h). A maior parte dos ncRNAs intrônicos possui estrutura cap 5\', sugerindo que sejam estabilizados para desempenhar papéis na biologia da célula que não dependam de um rápido turnover. A maior parte dos ncRNAs intrônicos é exportada para o citoplasma, indicando que devam exercer alguma função biológica neste compartimento. Em conjunto, este trabalho fornece informações novas a respeito da biogênese, estabilidade e localização sub-celular ncRNAs intrônicos expressos em células humanas, contribuindo para avançar o conhecimento sobre esta classe de transcritos celulares. / Recent studies have shown that most of the mammalian transcriptome is comprised of non-coding RNAs (lncRNAs). Our group has identified and characterized long (>200 nt), unspliced lncRNAs expressed in intronic regions of protein coding genes. However, the biogenesis, processing, stability and subcellular localization of members from this RNA class remain unknown. The aims of this work were i) to investigate the contribution of RNA Polymerase II (RNAP II) to the transcription of intronic, ii) to evaluate the half-life of these ncRNAs relative to mRNAs, and iii) determine their subcellular distribution. Our results indicate that intronic ncRNAs are predominantly transcribed by RNAP II from promoter regions functionally similar to those that control the transcription of mRNAs. Stability assays revealed that intronic ncRNAs have an average half-life equal or greater (3.4h to 4.2h) than mRNAs (3.1h). The majority of intronic ncRNAs have 5\' cap modification suggesting that these transcripts are stabilized, possibly to exert roles in the biology of the cell that does not depend on a rapid turnover. Although intronic ncRNAs do not encode proteins, most of these transcripts are transported to the cytoplasm which indicates that they may perform some biological function in this compartment. Altogether, this study reveals with novel information regarding the biogenesis, stability and subcellular localization of intronic ncRNAs expressed in human cells, thus contributing to advance the knowledge on this class of cellular transcripts.

Trabalhos recentes indicam que a maior parte do transcriptoma de células de mamíferos é composto por RNAs não-codificadores de proteínas (ncRNAs). Nosso grupo tem identificado e caracterizado ncRNAs longos (>200 nt), sem splicing, expressos em regiões intrônicas de genes codificadores de proteína. Contudo, a biogênese, processamento e localização sub-celular desta classe de RNAs permanecem desconhecidos. Este trabalho teve como objetivos i) investigar a contribuição da RNA Polimerase II (RNAP II) na transcrição de ncRNAs intrônicos, ii) avaliar a meia-vida destes ncRNAs em relação a mRNAs, e iii) verificar a distribuição sub-celular de ncRNAs intrônicos. Os resultados obtidos indicaram que ncRNAs intrônicos são predominantemente transcritos pela RNAP II a partir de regiões promotoras funcionalmente semelhantes as que controlam a transcrição de mRNAs. Ensaios de estabilidade revelaram que, em média, ncRNAs intrônicos possuem meia-vida igual ou maior (3,4h a 4,2h) do que mRNAs (3,1h). A maior parte dos ncRNAs intrônicos possui estrutura cap 5\', sugerindo que sejam estabilizados para desempenhar papéis na biologia da célula que não dependam de um rápido turnover. A maior parte dos ncRNAs intrônicos é exportada para o citoplasma, indicando que devam exercer alguma função biológica neste compartimento. Em conjunto, este trabalho fornece informações novas a respeito da biogênese, estabilidade e localização sub-celular ncRNAs intrônicos expressos em células humanas, contribuindo para avançar o conhecimento sobre esta classe de transcritos celulares. / Recent studies have shown that most of the mammalian transcriptome is comprised of non-coding RNAs (lncRNAs). Our group has identified and characterized long (>200 nt), unspliced lncRNAs expressed in intronic regions of protein coding genes. However, the biogenesis, processing, stability and subcellular localization of members from this RNA class remain unknown. The aims of this work were i) to investigate the contribution of RNA Polymerase II (RNAP II) to the transcription of intronic, ii) to evaluate the half-life of these ncRNAs relative to mRNAs, and iii) determine their subcellular distribution. Our results indicate that intronic ncRNAs are predominantly transcribed by RNAP II from promoter regions functionally similar to those that control the transcription of mRNAs. Stability assays revealed that intronic ncRNAs have an average half-life equal or greater (3.4h to 4.2h) than mRNAs (3.1h). The majority of intronic ncRNAs have 5\' cap modification suggesting that these transcripts are stabilized, possibly to exert roles in the biology of the cell that does not depend on a rapid turnover. Although intronic ncRNAs do not encode proteins, most of these transcripts are transported to the cytoplasm which indicates that they may perform some biological function in this compartment. Altogether, this study reveals with novel information regarding the biogenesis, stability and subcellular localization of intronic ncRNAs expressed in human cells, thus contributing to advance the knowledge on this class of cellular transcripts.