The assembly of the ribosome is a complex process that needs to be highly efficient to support maximum growth. Although the individual subunits of the ribosome can be reconstituted in vitro, such a reaction is inefficient in comparison to the assembly rate in vivo. What differentiates the in vivo from the in vitro assembly is primarily the presence of ribosome assembly proteins. These are proteins that assist in the assembly of the ribosomal subunits but are not part of the mature ribosome. In bacteria, the ribosome assembly proteins include rRNA processing enzymes and rRNA/ribosomal protein (r-protein) modifying enzymes. One set of ribosome assembly proteins, the ribosome maturation factors, have been difficult to classify due to their differences in structure and their apparent lack of similarities with regard to function. As part of this thesis, the previously uncharacterized RimP (ribosome maturation) protein formerly known as P15A or YhbC, was studied. Deletion of the rimP gene affected the growth rate more severely at 44°C than at 37°C and 30°C. Polysome profile analysis revealed a decrease in the amount of translating ribosomes and a corresponding increase in the amount of free 50S and 30S ribosomal subunits. The disproportionate large increase in 50S relative to 30S subunits indicated a 30S assembly defect. RimP was shown to localize to the 30S ribosomal subunit, and an accumulation of 17S rRNA, a precursor to 16S rRNA, supports a role for RimP in 30S subunit maturation. The results from in vitro reconstitution experiments have given valuable insights in the assembly of the 30S subunit. By using a recently developed method, the role of ribosome maturation factors Era, RimM and RimP during in vitro reconstitutions of the 30S subunit was investigated. Era was found to increase the incorporation rate for most of the late binding r-proteins, while RimM and RimP had more specific effects. RimM increased the incorporation rate for r-proteins S19 and S9 and inhibited the incorporation of S13 and S12, whereas RimP increased the incorporation rate primarily for S12 and S5. A comparison of the ribosome maturation factors RimP and RbfA (ribosome binding factor A) revealed structural similarities between the N-terminal domain of RimP and the single domain of RbfA. RbfA is a 15 kDa protein that was found to high copy-suppress a dominant C23U 16S rRNA mutation giving rise to cold-sensitivity in E. coli. A number of chromosomal suppressor mutations that increased the growth rate of an rbfA null mutant were isolated. The five strongest suppressor mutations were localized to the rpsE gene, for r-protein S5 and resulted in amino acid substitutions in three positions: G87A, G87S, G91A, A127V and A127T. These alterations improved translation and the processing of 16S rRNA in the rbfA null mutant. Moreover, they also suppressed the slow growth of the C23U rRNA mutant at 30, 37 and 44°C. / Monteringen av ribosomen är en komplex process som måste vara effektiv för cellen skall kunna växa så fort som möjligt. Det är visat sedan tidigare att ribosomens två subenheter kan monteras ihop in vitro och sedan vara del av en ribosom som gungerar vid proteinsyntes, dock är den typen av rekonstrueringsreaktioner mycket ineffektiva i jämförelse med vad som krävs in vivo. Skillnaden mellan dessa två tillstånd är primärt in vivo-reaktionens närvaro av hjälpproteiner. Hjälpproteinerna assisterar monteringen av ribosomens subenheter men är själva inte en del av den färdiga ribosomen. Inom denna klass av proteiner återfinns proteiner som t.ex. processerar ribosomalt RNA och proteiner som modifierar ribosomalt RNA och ribosomala protein. En klass av hjälpproteiner, mognadsfaktorerna, har varit svåra att klassificera på grund av strukturella olikheter och en brist på funktionella likheter. En del i detta avhandlingsarbete var karaktäriseringen av den tidigare okända mognadsfaktorn RimP, tidigare kallad YhbC eller P15A. En deletion av rimP hade störst påverkan på tillväxthastigheten vid 44°C, men effekter kunde även ses vid 30°C och 37°C. En analys av den ribosomala statusen visade på en minskning av ribosomer aktiva i translation och en motsvarande ökning av fria 50S- och 30S-subenheter. Den oproportionerligt höga ökningen av fria 50S-subenheter, i relation till 30S-subenheter, indikerade att något var fel i monteringen av 30S-subenheten. RimP-proteinet återfanns lokaliserat till fria 30S-subenheter, och en ökning av omoget 16S ribosomalt RNA i en stam som saknar RimP stödjer dess roll i monteringen av 30S-subenheten. Rekonstrueringsexperiment In vitro har gett många värdefulla ledtrådar till hur 30S-subenheten monteras ihop. Genom att använda en nyligen utvecklad metod kunde vi undersöka hur mognadsfaktorerna Era, RimM och RimP påverkade monteringen av ribosomens 30S subenhet in vitro. Era ökade inkorporeringshastigheten av många av de ribosomala proteiner som inkorporeras sent i monteringen av 30S, medans RimM och RimP uppvisade mer specifika effekter. RimM ökade inkorporeringshastigheten för de ribosomala proteinerna S19 och S9, men dessutom inhiberade RimM inkorporeringen av de ribosomala proteinerna S13 och S12. RimP uppvisade den tydligaste effekten av de undersökta proteinerna genom att kraftigt öka 8 inkorporeringshastigheten för det ribosomala proteinet S12, och ökade även inkorporeringshastigheten för det ribosomala proteinet S5. En jämförelse av de två mognadsfaktorerna RbfA och RimP visade på strukturella likheter mellan RimP:s N-terminala domän och den enda domänen hos RbfA. RbfA är ett 15 kDa protein som upptäcktes som en hög-kopiesupressor av en dominant C23U-mutation i 16S ribosomalt RNA som leder till köld-känslighet hos E. coli. Ett antal kromosomala supressormutationer som ökade tillväxthastigheten för en mutant som saknar RbfA isolerades och de fem starkaste av dessa lokaliserades till rpsE genen som kodar för det ribosomala proteinet S5. Mutationerna gav upphov till aminosyra utbyten i tre positioner i S5: G87A, G87S, G91A, A127T och A127V. Förändringarna i S5 förbättrade translationen och processningen av 16S ribosomalt RNA i mutantensom saknar RbfA. Dessutom förbättrade mutationerna tillväxthastigheten hos C23U-mutanten vid 30, 37 och 44°C.

La traduction est un processus itératif réalisé par le ribosome. Chez Escherichia coli, le ribosome est composé d’une grande sous-unité 50S et d’une petite sous-unité 30S (S correspondant au coefficient de sédimentation). La traduction débute par la mise en place d’une interaction entre le codon d’initiation de l’ARNm et l’anticodon de l’ARNt initiateur. Cette interaction, finement régulée par les facteurs d’initiations IF1, IF2 et IF3, conduit à la formation du complexe d’initiation 30S (30SIC). Par titration calorimétrique isotherme (ITC), nous avons disséqué la thermodynamique de l’ensemble des voies possibles de formation du 30SIC. Sur la base des affinités mesurées, il a été possible d’en déduire un ordre d’assemblage préférentiel. Par cryo-microscopie électronique, nous avons ensuite essayé d’obtenir la structure de ce complexe à haute résolution. La fixation du 50S représente la dernière étape de l’initiation. Par ITC, nous avons cherché à en déterminer les paramètres thermodynamiques, puis nous avons poursuivi avec l’élongation en commençant par étudier l’incorporation d’un aminoacyl-ARNt. Enfin, la réalisation d’une étude comparative par ITC de trois antibiotiques capables de se fixer au tunnel de sortie du peptide, nous a permis d’identifier les forces moléculaires mises en jeu lors de leur interaction avec le ribosome. / Translation is an iterative process achieved by the ribosomal machinery. In Escherichia coli, the ribosome is composed of a large 50S subunit and a small 30S subunit (S being the sedimentation coefficient). Translation begins with the establishment of the interaction between the mRNA codon and the initiator tRNA anticodon. This interaction, under the control of initiation factors IF1, IF2 and IF3, leads to the formation of the 30S initiation complex (30SIC). Using isothermal titration calorimetry (ITC), we explored the thermodynamic landscape of all possible pathways for 30SIC formation. Based on affinities derived from ITC, we propose a preferred assembly pathway. Using cryo-electron microscopy, this knowledge was used to obtain high-resolution structures of 30SIC intermediates. Binding of the 50S is the last step for initiation. Using ITC, thermodynamic parameters were derived followed by the incorporation of an aminoacyl-tRNA. Lastly, we realized, using ITC, a comparative study of three antibiotics binding to the nascent peptide exit tunnel of the ribosome. This study leads us to determine the molecular forces involved in their interaction with the ribosome.

Antibacterial agents specific for the 50S ribosomal subunit not only inhibit translation but also prevent assembly of that subunit. I examined the 30S ribosomal subunit in growing Escherichia coli cells to see if antibiotics specific for that subunit also had a second inhibitory effect. I used the aminoglycoside antibiotics paromomycin and neomycin, which bind specifically to the 30S ribosomal subunit. Both antibiotics inhibited the growth rate, viable cell number, and protein synthesis. I used a 3H-uridine pulse and chase assay to examine the kinetics of ribosome subunit assembly in the presence and absence of each antibiotic. Analysis revealed a concentration dependent inhibition of 30S subunit formation in the presence of each antibiotic. Sucrose gradient profiles of cell lysates showed the accumulation of an intermediate 21S translational particle. Taken together this data gives the first demonstration that 30S ribosomal subunit inhibitors can also prevent assembly of the small subunit.

The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E.coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio labeled antibiotics both in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug in vivo. In addition, cell free reconstitution assays generated a 21S particle during incubation with either aminoglycoside. Precursor formation was inhibited with increasing drug concentration. This work examines features of a novel antibiotic target for aminoglycoside and will provide information that is needed for the design of more effective antimicrobial agents.

<p>The ribosome is part of the indispensable machinery of every living cell. This large macromolecule, which decodes messenger RNA to produce proteins, is the subject of intense study as the mediator of an essential process. The prokaryotic ribosome is a major target for antimicrobial therapy, as its structure differs significantly from the eukaryotic ribosome. At present, the in vivo process of translation on the mature bacterial, or 70S, ribosome is well studied and increasingly understood, while the process of assembling the small (30S) and large (50S) subunits of this complex ribonucleoprotein enzyme has mostly been studied in vitro. Consequently, the significance of in vivo events such as ribosomal RNA (rRNA) maturation and factor-mediated maturation is incompletely understood. By studying the nature and structure of an in vivo assembled immature 30S subunit, this thesis aims to gain a better understanding of the key events in 30S subunit biogenesis. Deletion of the assembly cofactor Ribosome Maturation Factor M (RimM) results in slow growth, inefficient rRNA processing, and accumulation of nonfunctional, immature 30S subunits. This work presents the first cryo-EM model of the immature 30S purified from a RimM knockout strain of <em>E. coli</em>. The structure reveals distortion of the decoding centre and a disrupted 50S-binding interface, attesting to the importance of rRNA processing in 30S maturation. Additionally, the model suggests consequences for ribosomal protein incorporation and rRNA domain position relative to the mature 30S.</p> / Master of Science (MSc)

<p>Our understanding of the mechanism of ribosome assembly in bacteria is still in its infancy. Work from our laboratory and others have recently established that some protein assembly factors assist the assembly process at its late stages, mediating the correct folding of the functional core of the 30S and 50S subunits. The GTPase YjeQ is an assembly factor that displaces the upper domain of h44 of the mature 30S subunit upon binding, inducing a distortion in the decoding center. We hypothesized that the displacement of h44 is caused by the zinc-finger domain of YjeQ and mediates the release of RbfA, another assembly factor involved in 30S subunit maturation. To understand how the zinc-finger domain of YjeQ implements the functional interplay with RbfA, we constructed several deletion mutants of the domain. We found that the zinc-finger domain of YjeQ was required to bind the 30S subunit, but not the C-terminal extension (CTE) of the domain. The CTE was necessary for stimulation of GTPase activity upon binding to the 30S subunit and removal of bound RbfA from the 30S subunit. The data presented here suggests that the zinc-finger domain is essential for YjeQ to bind the 30S subunit and to implement the functional interplay with RbfA. Ongoing structural studies of the complex formed by the YjeQ CTE variant and the 30S subunit will provide a three dimensional view of the conformational changes that occur to implement the functional interplay between YjeQ and RbfA at the late stages of 30S subunit assembly.</p> / Master of Science (MSc)

<p>Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein.</p><p>Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated <i>Escherichia coli</i> individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ.</p><p>Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA.</p><p>We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD⁺or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.</p>

Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein. Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated Escherichia coli individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ. Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD+ or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.

This diploma thesis focuses on particular villas built in South Bohemian region at the beginning of the 20th century. The introductory chapters of this thesis explain the term "villa" and also briefly describe this specific type of construction. Follows the chapter summarising the most important architectural styles of the 20th century which is followed by the chapter concerning origin and development of functionalism both in the Czech Republic and in the rest of the world. The sixth chapter includes the core of this thesis by analysing particular South Bohemian villas. At this placethe author is focusing on personalities of the builders, architects hand in hand with a description of the original appearance of the buildings and their current state. The text of this thesis is for illustration amended by number of picture attachements.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A efervescência cultural e política dos anos 20 contribuiu para o surgimento do romance nordestino de 30. Nesse momento, o engajamento do intelectual era uma necessidade e as produções artísticas foram mediadas por influências políticas de esquerda ou de direita. Com isso, as obras literárias ganharam um tom de denúncia, expondo nossos problemas sociais. O intelectual brasileiro se viu entre dúvidas e tensões e destacamos, na primeira parte desse trabalho, as posturas dos seguintes escritores: José Américo de Almeida (1887-1980), Rachel de Queiroz (1910-2003), Jorge Amado (1912-2001), José Lins do Rego (1901-1957) e Graciliano Ramos (1892-1953). Na segunda parte, tomamos as obras do escritor paraibano, José Lins do Rego, para analisar o posicionamento desse intelectual frente as questões de sua época. Com base nas discussões que o escritor põe em relevo em seus romances, identificamos a abordagem da relação entre intelectual e operário, os limites da figuração do outro o negro e a mulher pobre , a continuidade e a ampliação da escravidão no século XX. Cotejando as respostas de José Lins com as dos demais romancistas nordestinos de 30, verificamos como se estabeleceu o diálogo entre esses escritores e quais são os limites de seus posicionamentos / The political and cultural effervescence of the roaring 20s contributed for the rising of the northeasterner literature romances of the 30s. In those days, the intellectual political engagement was a need and, therefore, influencing art productions which conveyed political issues embraced by either right and left wings of the Brazilian society. Consequently, literature works asssumed a denounce attitude by exposing our social issues.The Brazilian literate one was caught among doubts and social tensioned struggles, therefore emerging the following intellectual personalities of the time: José Américo de Almeida (1887-1980), Rachel de Queiroz (1910-2003), Jorge Amado (1912-2001), José Lins do Rego (1901-1957) and Graciliano Ramos (1892-1953) whose stands are covered in the first part of this work. The second part takes the works written by José Lins do Rego in order to analyze the positioning of this writer towards the social affairs ot that period. By this token, we approach the debates highlighted by that author in his books . To identify the ways he took so as to deal with the boundaries of his featured characters - such as the black person and the poor woman, the continued slavery and its entangled widening over the 20th century. By comparing the answers given by José Lins in discussions held with other authors in the 30s, one spots the dialog among these writers, their standpoints and the limits surrounding them