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Chiu, Yu Hin Teddy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Content --- p.vi / Abbreviations --- p.x / Naming system for mutant proteins --- p.xi / Abbreviation for amino acid --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- What are acidic ribosomal proteins? --- p.1 / Chapter 1.2 --- Why P-proteins are so important? --- p.13 / Chapter 1.3 --- Research objectives --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- List of buffers and media --- p.17 / Chapter 2.1.1 --- Preparation of buffers and media --- p.17 / Chapter 2.1.2 --- Buffers for preparing competent cells --- p.17 / Chapter 2.1.3 --- Media for bacterial culture --- p.17 / Chapter 2.1.4 --- Buffers for nucleic acid electrophoresis --- p.19 / Chapter 2.1.5 --- Buffers for protein electrophoresis --- p.19 / Chapter 2.1.6 --- Buffers for interaction studies using BIAcore 3000 --- p.21 / Chapter 2.2 --- General methods --- p.23 / Chapter 2.2.1 --- Preparation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.2 --- Transformation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.3 --- DNA cloning --- p.24 / Chapter 2.2.3.1 --- DNA cloning by polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.3.2 --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.2.3.3 --- Extraction and purification of DNA from agarose gels --- p.25 / Chapter 2.2.3.4 --- Restriction digestion of DNA --- p.25 / Chapter 2.2.3.5 --- Ligation of digested insert and expression vector --- p.27 / Chapter 2.2.3.6 --- Verification of insert by PCR --- p.27 / Chapter 2.2.3.7 --- Mini-preparation of plasmid DNA --- p.28 / Chapter 2.2.4 --- Polyacrylamide gel electrophoresis (PAGE) of protein --- p.29 / Chapter 2.2.4.1 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.4.2 --- Tricine SDS-polyacrylamide gel electrophoresis --- p.30 / Chapter 2.2.4.3 --- Native polyacrylamide gel electrophoresis --- p.31 / Chapter 2.2.4.4 --- Commassie brilliant blue staining of proteinin polyacrylamide gel --- p.32 / Chapter 2.2.4.5 --- Zinc Imidazole staining of protein in polyacrylamide gel --- p.33 / Chapter 2.2.5 --- Protein concentration determination --- p.33 / Chapter 2.2.6 --- Expression of recombinant proteins --- p.33 / Chapter 2.2.6.1 --- Expression of recombinant proteins using LB --- p.33 / Chapter 2.2.6.2 --- Expression of recombinant proteins using minimal medium --- p.34 / Chapter 2.2.6.3 --- Harvest and lysis of bacterial cell culture --- p.34 / Chapter 2.3 --- Protein purification --- p.36 / Chapter 2.3.1 --- Purification of ribosomal protein P1 and its deletion mutants --- p.36 / Chapter 2.3.1.1 --- Purification of P1 --- p.36 / Chapter 2.3.1.2 --- Purification of P1ΔC25 --- p.36 / Chapter 2.3.1.3 --- Purification of HisMBP-P1ΔC40 and HisMBP-P1ΔC47 --- p.37 / Chapter 2.3.2 --- Purification of ribosomal protein P2 and its deletion mutants --- p.38 / Chapter 2.3.2.1 --- Purification of P2 --- p.38 / Chapter 2.3.2.2 --- Purification of P2ΔC46 and P2ΔC55 --- p.39 / Chapter 2.4 --- "Preparation and purification of protein complexes formed by P1, P2 and their truncation mutants" --- p.40 / Chapter 2.4.1 --- Preparation of complexes by Co-refolding in urea buffer --- p.40 / Chapter 2.4.1.1 --- Preparation of P1 or P1ΔC25 involved complexes --- p.40 / Chapter 2.4.1.2 --- Preparation of P1ΔC40/ P2ΔC46 and P1ΔC47/ P2ΔC46 --- p.41 / Chapter 2.4.2 --- Preparation of complexes by direct mixing --- p.42 / Chapter 2.5 --- Laser light scattering for the determination of molecular weight of protein and their complexes --- p.43 / Chapter 2.5.1 --- Chromatography mode light scattering experiment (SEC/LS) --- p.43 / Chapter 2.6 --- Interaction study of P1 and P2 using BIAcore 3000 surface plasmon resonance (SPR) biosensor --- p.45 / Chapter 2.6.1 --- Immobilization of P2 onto CM5 sensor chips --- p.45 / Chapter 2.6.2 --- Kinetic measurements of P1 and P2 interaction --- p.46 / Chapter Chapter 3 --- Determination of domain boundaries for dimerization of P1/P2 --- p.46 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Preparation of P1，P2 and their truncation mutants --- p.50 / Chapter 3.2.1 --- Construction of P1 and P2 N-terminal domains (NTDs) --- p.50 / Chapter 3.2.2 --- P1 and its truncation mutants were purified in denaturing condition --- p.53 / Chapter 3.2.3 --- "P2, P2AC46 and P2AC55 were purified" --- p.56 / Chapter 3.3 --- Formation of complexes from P1，P2 and their truncation mutants --- p.59 / Chapter 3.3.1 --- "P1, P2 and their truncation mutants interact to yield protein complexes" --- p.49 / Chapter 3.3.2 --- P1AC47/P2AC46 is the smallest N-terminal domain complex --- p.63 / Chapter 3.4 --- Perturbation of P2 NTD upon binding with P1 --- p.65 / Chapter 3.4.1 --- "1H, 15N 一 HSQC spectrum of P2AC46 changed significantly upon binding with P1" --- p.65 / Chapter 3.4.2 --- P1/P2AC46 prepared by co-refolding and direct mixing give the same HSQC spectra --- p.66 / Chapter 3.5 --- Discussion --- p.69 / Chapter Chapter 4 --- Stochiometry of P1/P2 Complex is revealed by Light scattering --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- P1 and P2 interact in 1:1 molar ratio --- p.77 / Chapter 4.2.1 --- Purified P2 exists as homo-dimer in solution --- p.77 / Chapter 4.2.2 --- The stochiometry of P1/P2 complex is 1:1 --- p.78 / Chapter 4.3 --- Stochiometries of P1 and P2 truncation mutant complexes varied from the full-length counterparts --- p.81 / Chapter 4.3.1 --- P2AC46 and P2AC55 exist as homo-dimer in solution --- p.81 / Chapter 4.3.2 --- "P1/P2AC46, P1AC25/P2 and P1AC40/P2AC46 retain the hetero-dimeric stochiometry of 1:1" --- p.82 / Chapter 4.3.3 --- P2AC55 involved complexes show a different stochiometry --- p.83 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- Binding kinetics of P1/P2 complex studied by surface plasmon resonance --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Kinetic parameters of P1 and P2 interaction is revealed by surface plasmon resonance --- p.95 / Chapter 5.2.1 --- P2 was coupled to CM5 sensor chip surface for kinetic studies --- p.95 / Chapter 5.2.2 --- Reduction of basal response after the 1st binding of P1 --- p.96 / Chapter 5.2.3 --- P1 induced a great change in response unit than P2 upon binding with immobilized P2 --- p.99 / Chapter 5.2.4 --- Kinetic parameters of P1 and P2 interaction was studied by introducing P1 to the sensor chip surface --- p.101 / Chapter 5.2.5 --- Dissociation constant derived from 1:1 Langmuir binding isotherm --- p.102 / Chapter 5.2.6 --- Dissociation constant derived from responses at equilibrium (Req) --- p.103 / Chapter 5.3 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion and discussion of the study --- p.112 / References --- p.118 / Appendix --- p.130

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RESUME<p><p><p>Le proto-oncogène c-myc code pour un facteur de transcription qui est impliqué dans de multiples processus cellulaires tels que la prolifération, la différenciation et l’apoptose. Une dérégulation de son expression suite à des altérations génétiques (mutation, translocation, amplification) est retrouvée dans plusieurs tumeurs telles que le lymphome de Burkitt, des plasmacytomes murins ainsi que des tumeurs non-lymphoïdes.<p>c-myc est un gène dont l’expression est régulée à différents niveaux. Chez l’homme, le gène c-myc est transcrit à partir de quatre promoteurs alternatifs appelés respectivement P0, P1, P2 et P3. P1 et P2 sont les deux promoteurs les plus utilisés. Ensemble, ils permettent de former 90% des transcrits c-myc dans des cellules normales. <p>Les promoteurs P0, P1 et P2 permettent la transcription de trois ARNms qui comportent deux codons d’initiation de la traduction (un CUG et un AUG). L’utilisation alternative de ces deux codons d’initiation est à l’origine de la synthèse de deux protéines (c-Myc 1 et c-Myc 2) ayant à la fois des fonctions identiques et distinctes. <p> La grande taille des parties 5’ non-traduites ainsi que la présence dans celles-ci de phases ouvertes de lecture sont des éléments défavorables à la traduction de l’ORF codant pour les protéines Myc par un mécanisme classique d’initiation de la traduction. Notre laboratoire avait précisément montré que les protéines c-Myc sont synthétisées par un processus d’initiation interne de la traduction. Les ARNms dont l’initiation de la traduction s’effectue par entrée interne des ribosomes présentent une structure spécifique appelée IRES (Internal Ribosome Entry Site). Cette structure permet la fixation du ribosome directement à proximité du codon d’initiation. Dans le cas des ARNms c-myc, on retrouve une IRES se situant en amont des codons CUG et AUG qui permet la synthèse des protéines c-Myc1 et 2 respectivement. Un tel mécanisme permet la synthèse des protéines c-Myc dans des conditions où toute traduction dépendante de la coiffe est inhibée (mitose, apoptose).<p><p>Au cours de mon travail, tout d’abord j’ai montré qu’une séquence de 40 nt dans les transcrits P2 permet à elle seule une initiation interne efficace de la traduction. Nous avons déterminé aussi que cette séquence, appelée B4, est active dans quatre types cellulaires différents avec une efficacité variable et qu’elle active la traduction indépendamment de l’ORF placée en aval. D’autre part, il a été déterminé que la séquence B4 recrute le complexe de préinitiation 43S, qui ensuite scanne le messager jusqu’aux codons initiateurs comme c’est le cas de l’IRES du rhinovirus. <p>Une analyse plus détaillée de la séquence B4 a permis d’identifier trois plus petites séquences de plus ou moins 14 nt (Ti1, Boucle, Ti2), qui indépendamment l’une de l’autre permettent une entrée interne des ribosomes. Il a été déterminé que la présence du motif A-N6-AC dans la séquence de Ti2 était importante pour l’activité IRES de celle-ci. Cependant, ce même motif également présent dans la séquence Ti1 n’est pas essentiel à l’activité IRES de Ti1. <p>Par la suite, nous avons démontré que l’IRES de c-myc nécessite pour son activité un évènement nucléaire. Nous avons donc entrepris la recherche de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc. Dans un premier temps, nous avons exclu le rôle de certaines protéines connues pour activer d’autres IRES dont le mécanisme de recrutement du complexe de préinitiation est similaire. Ainsi, nous avons montré, par des expériences de complémentation d’un RRL, que les protéines PTB et unr connues pour activer l’IRES du rhinovirus ne contribuent pas à l’activité de l’IRES de c-myc. De plus, la complémentation de RRL avec des extraits S10 ou nucléaires de cellules HeLa n’a pas permis d’identifier des protéines impliquées dans l’activité IRES de c-myc.<p>D’autre part, des méthodes alternatives d’interaction d’ARN et de protéine comme le triple hybride ou la chromatographie d’affinité d’ARN n’a pas permis dans un premier temps de détecter une interaction entre un facteur non canonique et l’IRES de c-myc. Dès lors, l’existence de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc reste à déterminer.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

Cryo-electron microscopy (cryo-EM) is a structural biology technique that determines the structure of proteins and macromolecular complexes using the transmission electron microscope under cryogenic conditions. In my Ph.D. studies, I took advantage of this technique, in the study of dynamical features of ribosomes in both eukaryotes and prokaryotes. In Chapter 2, I report my graduate research on the investigation of ribosomes from the human malaria parasite, Plasmodium falciparum, using single-particle cryo-EM. In collaboration with Dr. Jeffrey Dvorin at Harvard Medical School, we obtained five cryo-EM reconstructions of ribosomes purified from P. falciparum blood-stage schizonts, and discovered structural and dynamical features that differentiate the ribosomes of P. falciparum from those of the mammalian system. Moreover, we discovered that RACK1, a necessary ribosomal protein in eukaryotes, does not specifically co-purify with the 80S fraction in the P. falciparum schizonts stage and would mainly function in a ribosome-unbound, free state during the blood-stage. More extensive studies, using cryo-EM methodology, of translation in the parasite, will provide structural knowledge that could help in the design of effective anti-malaria drugs. In Chapter 3, I describe the cryo-EM studies of the Saccharomyces cerevisiae ribosome in response to a carbon source switch. In collaboration with Dr. Andrew Link at Vanderbilt University, we obtained reconstructions of the 80S ribosomes at selected time points after the glucose-to-glycerol carbon source shift, and observed that a fraction of ribosomes lacked densities for r-proteins, mainly eS1 (yeast rpS1) on the 40S subunit and uL16 (yeast rpL10) on the 60S subunit. We found that the binding ratio of eS1 and uL16 to ribosomes changed as a function of time, consistent with the change in translational activities as gauged by polysome profiling. On the basis of these observations, along with previous structural and genetics studies, we propose that rapid control of translation is exerted through the dissociation of r-protein eS1/rpS1 and uL16/rpL10 from the ribosome. Our studies thus open a new venue on the exploration of S. cerevisiae’s rapid adaption to carbon source shifts at the level of translation. In Chapter 4, I have documented a collaborative work on the development and application of a new technique, time-resolved cryo-EM, which can be used to study processes involving two reaction partners on a sub-second time scale. With my colleagues at the Frank and Gonzalez labs at Columbia University, we successfully applied this method to study the process of E. coli ribosomal subunits association. By mixing and reacting the two subunits for 60 ms and 140 ms, we captured the association reaction in a pre-equilibrium state, and detected different conformations of E. coli 70S ribosomes. With the current capability of this mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, we expect to be able to standardize this method and apply it to more challenging biological processes, such as translation recycling and initiation processes.

The translational GTPases elongation factor Tu (EF-Tu) and LepA modulate the dynamics of tRNA on the ribosome. EF-Tu facilitates the delivery of aminoacyl-tRNA (aa-tRNA) to the translating ribosome and LepA catalyzes the retro-translocation of tRNA•mRNA from the E- and P-sites of the ribosome back to the P- and A-sites. Although an increasing body of structural and biochemical information is available, little is known about the functional cycle of LepA during retro-translocation, the kinetics of EF-Tu dissociation from the ribosome and the rate of EF-Tu conformational change during aa-tRNA delivery. This thesis reports the successful construction and biochemical characterisation of a mutant form of EF-Tu from Escherichia coli ideal for the specific incorporation of fluorescent labels, enabling measurements pivotal for uncovering the rate of EF-Tu conformational change and dissociation from the ribosome. Furthermore, to determine structural components critical for LepA’s function, mutant versions of the protein were constructed and biochemically characterised. / xii, 127 leaves : ill. (some col.) ; 29 cm

Six ribosomal protein genes from the sulfur dependent extreme thermophilic archaebacterium Sulfolobus solfataricus were cloned and sequenced. Four of these genes code for proteins that are equivalent to ribosomal proteins L11, L1, L10 and L12 in Escherichia coli. The other two genes code for proteins that have no equivalent in the eubacteria. The product of one of these genes was found to be equivalent to ribosomal proteins L46 from yeast (Leer et al. 1985a) and L39 from rat liver (Lin et al. 1984), while the product of the other gene shows no sequence similarity to any of the ribosomal proteins present in the data base. In Sulfolobus, the genes that code for ribosomal proteins L11, L1, L10 and L12 are organized in the same order as in Escherichia coli, that is 5' L11, L1, L10, L12 3'. The major transcript from this gene cluster was found to be a 2.5 Kb mRNA that contains the four genes. A less abundant transcript containing only the L10 and L12 gene was also detected. Upstream of the transcription initiation sites, sequences that match the consensus sequence for archaebacterial promoters (TTTAT/AA) were found. Transcription termination sites were located within or after pyrimidine rich regions. Three of the ribosomal protein genes start with unusual initiation codons, GTG in the case of the L1 and L10 genes and TTG in the case of the L11 gene. Putative Shine Dalgarno sequences, complementary to the 3' end of Sulfolobus 16S rRNA, were detected in the region surrounding the initiation codon. In some cases (L1 and L10 genes), the initiation codon was found to be part of this sequence. Sequence comparison of the ribosomal proteins from Sulfolobus with those from other organisms, revealed that the Sulfolobus sequences are closer to those from other archaebacteria, thus supporting the existence of the archaebacterial kingdom. Comparison of the sequences of the L10 and L12 proteins from the three kingdoms revealed that the archaebacterial sequences are closer to the eukaryotes. / Graduate

Eukaryotic ribosome synthesis is a complex process that consumes a lot of energy and involves several hundreds of trans-acting factors that transiently associate with nascent ribosomes. Biogenesis of ribosomal subunits (the small 40S and the large 60S) starts with transcription of a long precursor ribosomal RNA (pre-rRNA) by RNA polymerase I (Pol I) in the nucleolus. This is a key step that globally controls yeast ribosome synthesis. The pre-rRNA, ‘the 35S transcript’, encodes the mature sequence (18S, 5.8S, and 25S) rRNA constituents of both the 40S and 60S subunits. The 35S transcript is subsequently modified, cleaved (processed) and assembled with numerous structural ribosomal proteins and ribosome synthesis factors (trans-acting factors) to form various ribosomal particles (pre-ribosomes, precursors to the 40S and 60S subunits) along ribosome assembly pathway. <p>In the budding yeast Saccharomyces cerevisiae, it has been reported recently that the processing of the 35S nascent transcript and the assembly of pre-ribosomes occur concomitantly with Pol I transcription in the nucleolus. In this process, the growing Pol I transcript gradually assembles with pre-40S structural ribosomal proteins and ribosomal synthesis factors to form the so-called ‘SSU-processome’ or ‘90S pre-ribosome’, the earliest precursor of the 40S subunit. The SSU-processome/90S pre-ribosome localizes to the nucleolus and consists of the 35S pre-rRNA, the U3 small nucleolar (sno) RNA, about a dozen of 40S ribosomal proteins and more than forty ribosome synthesis factors. The U3 snoRNA and pre-40S ribosome synthesis factors are all implicated in the processing of the 35S precursor (at sites A0, A1 and A2) and therefore in the synthesis of the 18S rRNA component of the 40S subunit. Significantly, the association of the U3 snoRNA with the growing 35S transcript is important for pre-40S assembly, whereas its dissociation from the processed transcript (following cleavage at sites A0-A2) is crucial for the overall structural remodeling of the 18S rRNA and for the formation of pre-40S ribosomes from the earliest precursor 90S particles. <p>This thesis mostly addresses the identification and functional characterization of Esf2 and Bfr2, two novel 40S synthesis factors, components of the SSU-processome/90S pre-ribosome in yeast. Both proteins localize to the nucleolus and their genetic depletions lead to failure in the production of 40S subunits. In the absence of either factor, the 35S pre-rRNA is not processed at sites A0-A2 and the 18S rRNA is not synthesized. Also, pre-ribosome assembly is affected and pre-40S ribosomes fail to mature properly. Strikingly, in the absence of either factor, the U3 snoRNA remains associated with unprocessed 35S transcript within pre-ribosomes indicating that Esf2 and Bfr2 are required to dissociate U3 from pre-ribosomes. This process also involves RNP (ribonucleoprotein particle) unwinding activities of the putative RNA helicase Dbp8. <p>La biogenèse du ribosome eucaryote est un processus complexe qui consomme beaucoup d’énergie et implique plusieurs centaines de facteurs trans qui s’associent de manière transitoire avec les pré-ribosomes en cours de formation. La biogenèse des sous-unités ribosomiques (la petite sous-unité 40S et la grande sous-unité 60S) débute dans le nucléole par la synthèse d’un long précurseur d’ARN ribosomique (le pré-ARNr, dit 35S chez la levure Saccharomyces cerevisiae) par l’ARN Polymérase I (Pol I). Ceci constitue une étape clé dans le contrôle global de la synthèse du ribosome chez la levure. Le pré-ARNr 35S renferme les séquences des ARNr matures 18S (ARNr de la sous-unité 40S) et 5.8S et 25S (deux des trois ARNr de la sous-unité 60S). Le pré-ARNr 35S subit un long processus de maturation et d’assemblage au cours duquel il est modifié, clivé (on parle du « processing » du pré-ARNr) et s’assemble avec des protéines ribosomiques (« RP », composants structuraux des sous-unités ribosomiques matures) et de nombreux facteurs de synthèse (facteurs trans) pour former différentes particules pré-ribosomiques (précurseurs des sous-unités 40S et 60S).<p><p>Chez la levure S. cerevisiae, il a récemment été montré que le processing du pré-ARNr 35S et l’assemblage des pré-ribosomes se produisent de manière concomminante avec la transcription Pol I dans le nucléole. Ainsi, le transcrit Pol I en cours de synthèse s’assemble progressivement avec des facteurs de synthèse ainsi que des RP pour former le « SSU processome » ou « pré-ribosome 90S », tout premier précurseur de la petite sous-unité 40S. Le SSU processome/pré-ribosome 90S est localisé dans le nucléole et est consisté du pré-ARNr 35S naissant, du petit ARN nucléolaire (snoRNA) U3, d’une douzaine de RP de la petite sous-unité 40S et de plus de 40 facteurs de synthèse. Le snoRNA U3 et ces facteurs de synthèse sont tous impliqués dans les clivages du pré-ARNr 35S aux sites A0, A1 et A2, et donc dans la biogenèse de l’ARNr 18S. L’association du snoRNA U3 avec le pré-ARNr 35S naissant est importante pour l’assemblage du SSU processome/pré-ribosome 90S. Par ailleurs, sa dissociation après les clivages aux sites A0-A2 permet un remodelage structural général de l’ARNr 18S et la formation du « pré-ribosome 40S » à partir de la particule précoce 90S.<p><p>Au cours de cette thèse, nous avons identifié et caractérisé fonctionnelement chez la levure deux nouveaux facteurs de synthèse de la petite sous-unité 40S et composants du SSU processome/pré-ribosome 90S: Esf2 et Bfr2. Ces deux protéines sont localisées dans le nucléole. Leur déplétion entraîne une incapacité à produire la sous-unité ribosomique 40S. En l’absence d’Esf2 ou Bfr2, le pré-ARNr 35S n’est plus clivé aux sites A0-A2 et l’ARNr 18S mature n’est plus produit. L’assemblage des pré-ribosomes est aussi affecté, notamment la formation du pré-ribosome 40S. De manière importante, en l’absence de l’un ou l’autre de ces facteurs, le snoRNA U3 reste associé au pré-ARNr 35S non clivé au sein des pré-ribosomes, indiquant qu’Esf2 et Bfr2 sont requises pour la dissociation d’U3 des pré-ribosomes. Ce processus implique aussi Dbp8, une hélicase à ARN présumée.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished