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1
Slinger, Betty L. "Insights into the Co-Evolution of Ribosomal Protein S15 with its Regulatory RNAs". Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:106793.
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Thesis advisor: Michelle M. MeyerRibosomes play a vital role in all cellular life translating the genetic code into functional proteins. This pivotal function is derived from its structure. The large and small subunits of the ribosome consist of 3 ribosomal RNA strands and over 50 individual ribosomal proteins that come together in a highly coordinated manner. There are striking differences between eukaryotic and prokaryotic ribosomes and many of the most potent antibacterial drugs target bacterial ribosomes (e.g. tetracycline and kanamycin). Bacteria spend a large amount of energy and nutrients on the production and maintenance of these molecular machines: during exponential growth as much as 40% of dry bacterial mass is ribosomes (Harvey 1970). Because of this, bacteria have evolved an elegant negative feedback mechanism for the regulation of their ribosomal proteins, known as autoregulation. When excess ribosomal protein is produced, unneeded for ribosome assembly, the protein binds a structured portion of its own mRNA transcript to prevent further expression of that operon. Autoregulation facilitates a quick response to changing environmental conditions and ensures economical use of nutrients. My thesis has investigated the autoregulatory function of ribosomal protein S15 in diverse bacterial phyla. In many bacterial species, when there is excess S15 the protein interacts with an RNA structure formed in the 5’-UTR of its own mRNA transcript that enables autoregulation of the S15-encoding operon, rpsO. For many ribosomal proteins (ex. L1, L20, S2) there is striking homology and often mimicry between the recognition motifs within the rRNA and the regulatory mRNA structure. However, this is not the case for S15-three different regulatory RNA structures have been previously described in E. coli, G. stearothermophilus, and T. thermophilus (Portier 1990, Scott 2001, Serganov 2003). These RNAs share little to no structural homology to one another, nor the rRNA, and they are narrowly distributed to their respective bacterial phyla, Gammaproteobacteria, Firmicutes, and Thermales. It is unknown which regulatory RNA structures control the expression of S15 outside of these phyla. Additionally, previous work has shown the S15 homolog from G. stearothermophilus is unable to regulate expression using the mRNA from E. coli. These observations formulate the crux of the question this thesis work endeavors to answer: What drove the evolution of such diverse regulatory RNA structures in these different bacteria? In Chapter II, “Discover and Validate Novel Regulatory Structures for Ribosomal Protein S15in Diverse Bacterial Phyla”, I present evidence for the in silico identification of three novel regulatory RNA structures for S15 and present experimental evidence that one of these novel structures is distinct from those previously described. In Chapter III, “Co-evolution of Ribosomal Protein S15 with Diverse Regulatory RNA Structures”, I present evidence that the amino acid differences in S15 homologs contribute to differences in mRNA binding profiles, and likely lead to the development of the structurally diverse array of the regulatory RNAs we observe in diverse bacterial phyla. In Chapter IV, “Synthetic cis-regulatory RNAs for Ribosomal Protein S15”, I investigate the derivation of novel cis-regulatory RNAs for S15 and find novel structures are readily-derived, yet interact with the rRNA-binding face of S15. Together the work presented in this thesis advances our understanding of the co-evolution between ribosomal protein S15 and its regulatory RNAs in diverse bacterial phyla
Thesis (PhD) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Girnary, Roseanne Waheeda. "Structural and functional studies of the stimulatory RNAs involved in programmed -1 ribosomal frameshifting and translational readthrough". Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612716.
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Huang, Hsiau-Wen. "Investigation of solution structures of yeast and lupin seed 5S ribosomal RNAs by high resolution nuclear magnetic resonance and molecular dynamics simulation /". The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487684245468516.
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Crandall, Jacob N. "Ribosomal RNA Mutations that Inhibit the Activity of Transfer-Messenger RNA of Stalled Ribosomes". Diss., CLICK HERE for online access, 2010. http://contentdm.lib.byu.edu/ETD/image/etd3535.pdf.
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Roy, Poorna. "Deconstructing the ribosome: specific interactions of a ribosomal RNA fragment with intact and fragmented L23 ribosomal protein". Thesis, Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/47579.
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The complexity of translation is a classical dilemma in the evolution of biological systems. Efficient translation requires coordination of complex, highly evolved RNAs and proteins; however, complex, highly evolved RNAs and proteins could not evolve without efficient translation system. At the heart of this complexity is the ribosome, itself a remarkably complex molecular machine. Our work illustrates the ribosome as deconstructed units of modification.
Here we have deconstructed a segment of the ribosome to interacting RNA-protein units. L23 interacts in vivo with both Domain III (DIII) and Domain IIIcore (DIIIcore) independently of the fully assembled ribosome. This suggests that DIIIcore represents the functional rRNA unit in DIII-L23 interaction. Furthermore, L23peptide sustains binding function in vitro with both DIII and DIIIcore independently of any stabilizing effects from the globular domain of L23. The ability of L23peptide to form a 1:1 complex with both DIII and DIIIcore suggests that L23peptide is the functional rProtein unit in DIII-L23 interaction. We believe that our results will stimulate interest and discussions in the significance of 3D architecture and units of evolution in the ribosome. The ubiquity of the ribosome in cellular life prognosticates that our results impact and appeal to biologists, chemists, bioinformaticists, as well as the general scientific community.
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Burlacu, Elena. "Probing ribosomal RNA structural rearrangements : a time lapse of ribosome assembly dynamics". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/17072.
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Ribosome synthesis is a very complex and energy consuming process in which pre-ribosomal RNA (pre-rRNA) processing and folding events, sequential binding of ribosomal proteins and the input of approximately 200 trans-acting ribosome assembly factors need to be tightly coordinated. In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus with the formation of a very large 90S-sized complex. This ~2.2MDa pre-ribosomal complex is subsequently processed into the 40S and 60S assembly intermediates (pre-40S and pre-60S), which subsequently mature largely independently. Although we have a fairly complete picture of the protein composition of these pre-ribosomes, still very little is known about the rRNA structural rearrangements that take place during the assembly of the 40S and 60S subunits and the role of the ribosome assembly factors in this process. To address this, the Granneman lab developed a method called ChemModSeq, which made it possible to generate nucleotide resolution maps of RNA flexibility in ribonucleoprotein complexes by combining SHAPE chemical probing, high-throughput sequencing and statistical modelling. By applying ChemModSeq to ribosome assembly intermediates, we were able to obtain nucleotide resolution insights into rRNA structural rearrangements during late (cytoplasmic) stages of 40S assembly and for the early (nucleolar) stages of 60S assembly. The results revealed structurally distinct cytoplasmic pre-40S particles in which rRNA restructuring events coincide with the hierarchical dissociation of assembly factors. These rearrangements are required to trigger stable incorporation of a number of ribosomal proteins and the completion of the head domain. Rps17, one of the ribosomal proteins that fully assembled into pre-40S complexes only at a later assembly stage, was further characterized. Surprisingly, my ChemModSeq analyses of nucleolar pre-60S complexes indicated that most of the rRNA folding steps take place at a very specific stage of maturation. One of the most striking observations was the stabilization of 5.8S pre-rRNA region, which coincided with the dissociation of the assembly factor Rrp5 and stable incorporation of a number of ribosomal proteins.
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Ramesh, Madhumitha. "Analysis of Ribosome Biogenesis from Three Standpoints: Investigating the Roles of Ribosomal RNA, Ribosomal Proteins and Assembly Factors". Research Showcase @ CMU, 2016. http://repository.cmu.edu/dissertations/609.
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Ribosomes are ubiquitous and abundant molecular machines composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). They play a central role in the cell by translating the genetic code in mRNA to form polypeptides. Because of their large size and the complexity of molecular interactions within ribosomes, we do not still fully understand how they are synthesized in the cell. Yet, a thorough knowledge of ribosome biogenesis is crucial to understand cellular homeostasis and various disease states including ribosomopathies and cancer. In addition, ribosomes serve as an interesting paradigm to understand the principles that dictate the formation and function of the many different ribonucleoprotein particles that play vital roles in the cell. In addition to the rRNA and r-protein components, trans-acting assembly factors play indispensable roles in synthesizing functional ribosomes. Fundamentally, ribosome biogenesis is driven by a network of molecular interactions that evolve in time and space, as assembly progresses from the nucleolus to the cytoplasm. We sought to gain a deeper understanding of ribosome biogenesis in Saccharomyces cerevisiae by investigating the molecular interactions that drive ribosome assembly. Recent structural studies have revealed a number of such molecular interactions at high resolution. Based on these, our investigation was carried out from the perspectives of all three players that are involved in constructing ribosomes, with a specific emphasis on eukaryote-specific elements of rRNAs and r-proteins. From the standpoint of rRNA, we performed the first systematic study to investigate the potential functions of nearly all of the eukaryotic rRNA expansion segments in the yeast large ribosomal subunit. We showed that most of them are indispensable and play vital roles in ribosome biogenesis. Based on the steps of ribosome biogenesis in which each of them participates, we showed that there is neighborhood-specific functional clustering of rRNA and r-protein interactions that drive ribosome assembly. Further, we found evidence for possible functional co-evolution of eukaryotic rRNA and eukaryote-specific elements of r-protein. From the standpoint of r-protein, we used rpL5 as a paradigm for constantly evolving molecular interactions as assembly progresses. Apart from recapitulating Diamond-Blackfan anemia missense mutations in yeast, we characterized interactions formed by specific regions of rpL5 and propose that these interactions potentially govern the loading of 5S RNP en bloc to the nascent large ribosomal subunit, to ensure proper rotation of the 5S RNP during biogenesis, and to further recruit proteins necessary for the test drive of subunits in the cytoplasm. From the standpoint of assembly factors, we analyzed a so-called group of ITS2 cluster proteins, Nop15, Cic1 and Rlp7 and identified the extensive protein-protein interactions and analyzed protein-RNA interactions that they make. Using our data, we were able to localize Rlp7 to the ITS2 spacer in the pre-rRNA and to identify potential mechanisms for their function. Having identified a network of molecular interactions, we suggest that these proteins orchestrate proper folding of rRNA through this network, and stabilize and facilitate the early steps of assembly. Further, based on their location in the preribosome, these factors might serve to ensure proper progression of early steps of assembly to enable subsequent processing of the ITS2 spacer in the middle steps, possibly by recruiting the ATPase Has1. Thus, we have investigated early nucleolar and late nuclear steps of ribosome assembly in the light of molecular interactions formed by rRNA, r-protein and assembly factors that participate in eukaryotic ribosome assembly. Lessons that emerged from this study and tools developed in the process provide a starting point for further investigations pertaining to the roles of eukaryote-specific segments of molecules that participate in ribosome biogenesis, and serve as a paradigm for how a dynamic network of molecular interactions can drive the assembly of complex macromolecular structures.
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Weaver, Paul L. "Characterization of a putative RNA helicase, Dbp3p, in ribosomal RNA processing and ribosome biogenesis in Saccharomyces Cerevisiae /". The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu148794750113696.
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G, C. Keshav. "Investigation of the Role of Bacterial Ribosomal RNA Methyltransferase Enzyme RsmC in Ribosome Biogenesis". Kent State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=kent1621868567263046.
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Kshetri, Man B. "N-TERMINAL DOMAIN OF rRNA METHYLTRANSFERASE ENZYME RsmC IS IMPORTANT FOR ITS BINDING TO RNA AND RNA CHAPERON ACTIVITY". Kent State University Honors College / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1621007414429417.
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Bouakaz, Elli. "Choice of tRNA on Translating Ribosomes". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6324.
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Huynh, Dung Minh. "Mapping pseudouridine sites in Homo sapiens 18S ribosomal RNA, and the 3 dimensional ribosome map of nucleotide modifications". Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402266.
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Wilson, Williamina. "Ribosomal frameshifting in retroelements". Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670301.
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Hadjeras, Lydia. "Localisation membranaire de la RNase E : rôle dans la dégradation des ARN et la biogenèse des ribosomes". Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30231.
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La RNase E chez Escherichia coli est une endoribonucléase essentielle qui joue un rôle important dans la maturation des ARN stables, dans le contrôle qualité des ribosomes, ainsi que dans la dégradation constitutive et régulée des ARN messagers. La séquence de ciblage à la membrane (MTS pour Membrane Targeting Sequence), qui forme une hélice α-amphipatique, ancre la RNase E à la membrane cytoplasmique interne des cellules. La conservation absolue du MTS chez l'ensemble des -protéobactéries suggère un rôle important de la localisation membranaire RNase E dans le métabolisme de l'ARN. Pour élucider la fonction cellulaire de l'association membranaire de la RNase E, nous avons caractérisé la souche rne∆MTS qui exprime une RNase E cytoplasmique. Les résultats de cette étude nous amènent à proposer que l'association membranaire de la RNase E est nécessaire à la stabilité de la RNase E, est impliquée dans des interactions fonctionnelles avec des régulateurs associés à la membrane et protège les transcrits présents dans le nucléoïde en évitant des interactions prématurées avec la RNase E. En particulier, garder la RNase E à la membrane est critique pour la spécificité de la RNase E dans le contrôle qualité des ribosomes. Cette association membranaire est une nouvelle couche de régulation qui permet d’expliquer comment la RNase E, une enzyme avec peu de spécificité de séquence et avec beaucoup de substrat, peut remplir les fonctions de «maturase» et de «dégradase»RNase E in Escherichia coli is an essential endoribonuclease with important roles in stable RNA maturation, in ribosome quality control and in constitutive and regulated mRNA degradation. The Membrane Targeting Sequence (MTS), which forms an amphipathic α-helix, anchors RNase E on the inner cytoplasmic membrane. The absolute conservation of the MTS among -Proteobacteria suggests an important role for RNase E membrane association in RNA metabolism. To elucidate the cellular function of the membrane association of RNase E, we characterized the rne∆MTS strain expressing cytoplasmic RNase E. The results of this study lead us to propose that RNase E membrane association is necessary for RNase E stability, for functional interactions with membrane-associated regulatory factors and for protecting nascent transcripts in the nucleoid from premature interactions with RNase E. In particular, keeping RNase E to the membrane is critical for the specificity of RNase E in ribosome quality control. Membrane association is a new layer of regulation that can explain how RNase E, an enzyme with little sequence specificity and many substrates, can fulfill both ‘maturase’ and ‘degradase’ functions
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Deiorio-Haggar, Kaila. "RNA structures regulating ribosomal protein biosynthesis". Thesis, Boston College, 2015. http://hdl.handle.net/2345/bc-ir:104628.
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Thesis advisor: Michelle M. MeyerMost commonly known for being the blueprint for proteins, RNA also plays vital roles in gene regulation. Non-coding RNAs, functional RNA molecules that are not translated into proteins, are potential regulatory agents in bacteria. Ribosomal autogenous regulatory elements are short transcribed sequences between the promoter and a protein coding region that regulate expression of their associated gene(s), though they are not themselves translated. These sequences form RNA secondary structures that can regulate at either the transcriptional or translational level. These riboregulators have been well characterized in gram-negative bacteria such as Escherichia coli, but in gram-positive bacteria far less is known regarding how r-proteins are regulated. My main goal has been to find riboregulators of r-protein synthesis in Bacilli and determine their consensus structures and phylogenetic distributions. I have utilized the RNA homology search program Infernal, coupled with our high-capacity genomic context visualization tool, to identify homologues of ribosomal-protein autogenous regulatory RNAs found in Bacilli. The alignments produced from this work determine consensus secondary structures and phylogenetic distribution of these regulator RNAs that provide new insight into the structure and function of these RNAs
Thesis (MS) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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MIYOSHI, Masaya, Tetsuya OKAJIMA, Tsukasa MATSUDA, Michiko N. FUKUDA y Daita NADANO. "Bystin in human cancer cells : intracellular localization and function in ribosome biogenesis". Biochemical Society, 2007. http://hdl.handle.net/2237/9306.
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Nathania, Lilian. "Biochemical Analysis of Thermotoga maritima Ribonuclease III and its Ribosomal RNA Substrates". Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/140013.
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ChemistryPh.D.
The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.
Temple University--Theses
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Chang, Jen-Chyi. "Ribosomal RNA gene spaacers in Trichophyton violaceum". Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418770.
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Smith, David. "Minor form of human 5.8s ribosomal RNA". Diss., Virginia Polytechnic Institute and State University, 1986. http://hdl.handle.net/10919/71189.
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Part One: The Minor Form of Human 5.8S rRNA
An elongated form of 5.8S rRNA has been found in a wide range of eukaryotes from yeast to rodents. This minor form of 5.8S rRNA is about six nucleotides longer than the major form and composes from 10% to 30% of the total 5.8S rRNA found in yeast and rodents respectively. The minor form of 5.8S rRNA (pCCGAUA-) found in mice and rats may be generated by the formation of a secondary cleavage site caused by heterogeneity in the rRNA genes. The insertion of an adenylic acid residue in the precursor rRNA generates this additional cleavage site, i.e. -ACGA- or -ACCGA for the major and minor forms respectively. There is also heterogeneity with respect to the degree of methylation in rodent 5.8s rRNA. The conformation of the two chain length isomers is influenced by 2'-O-methylation of the uridylic acid residue at position 14, i.e. the most compact conformation is not ribose methylated in that position. The molecules which are methylated in the 14th position cannot adopt the most compact conformation. In the present study I have discovered a minor form of 5.8S rRNA in human placenta and I have determined its sequence; it differs from the major form of human 5.8S rRNA in having an additional sequence (CUCGUA) on the 5'-terminus. The sequence of the major rodent 5.8S rRNA is completely conserved in the major human 5.8S rRNA but the elongation on the 5'-ends of the minor 5.8S rRNAs from the two species are only 50% conserved. Human minor 5.8S rRNA was completely methylated at the uridylic acid residue at 14 making it the first 5.8S rRNA found to be completely methylated.
Part Two: Purification and Characterization of a Ribose Transmethylase from Ehrlich Ascites Cells
A filter binding assay was developed for measuring ribose transmethylase activity in cell extracts and was used to quantify ribose and base transmethylase in Ehrlich ascites cells and normal mouse liver. Ribose and base transmethylase activities were elevated two-fold in Ehrlich ascites cells compared to normal mouse liver when methyl-deficient mouse tRNA was used as substrate but base transmethylase activity was elevated tenfold in Ehrlich ascites cells when E. coli tRNA was used as substrate. E. coli tRNA did not serve as a methyl acceptor for ribose transmethylases.
The ribose transmethylase was purified 910-fold from Ehrlich ascites cell extracts and complete elimination of base transmethylase was achieved in one experiment. This purified ribose transmethylase was found to have an apparent KmtRNA of 20uM tRNA and an apparent KmSAM of 12.8uM SAM. The apparent molecular weight of the ribose transmethylase, as determined by gel filtration chromatography, was 240, 000 daltons. SDS-PAGE of the purified ribose transmethylase showed a predominant protein band of approximately 60,000 daltons.Ph. D.
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Rohlfs, Rebecca L. "Mass Spectrometry Analysis of Methylated Ribosomal RNA". University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1368024445.
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Böhm, Stefanie. "Non-protein-coding RNA : Transcription and regulation of ribosomal RNA". Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-102718.
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Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript
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Akhtar, Y. "Studies on the maturation pathway of ribosomal precursor RNA : Analysis of Xenopus ribosomal RNA synthesised by transcription in vitro". Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382054.
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Grierson, Patrick Michael. "The BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1337865492.
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Burger, Kaspar. "CDK9 links RNA polymerase II transcription to processing of ribosomal RNA". Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-167037.
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Morello, Luis Gustavo 1982. "Caracterização funcional das proteínas NIP7 e FTSJ3 no processamento do RNA ribossomal em células humanas". [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317176.
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Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Estudos prévios realizados em nosso laboratório demonstraram a interação entre as proteínas humanas SBDS e NIP7. SBDS participa da biogênese de ribossomos e sua deficiência está associada à síndrome de Shwachman- Bodian-Diamond. NIP7 é uma proteína conservada e já foi caracterizada em levedura, onde participa da formação da subunidade ribossomal 60S. Neste trabalho, nós investigamos o papel de NIP7 na síntese de ribossomos em células humanas. A depleção de NIP7 revelou defeitos no processamento do pré-rRNA associado à produção do rRNA 18S, causando déficit na formação da subunidade ribossomal 40S. Essa divergência de resultados entre a função de NIP7 em levedura e células humanas é consistente com o fato de que NIP7 humana não complementa levedura deficiente em Nip7p. Ainda, um rastreamento em sistema de duplo-híbrido tendo NIP7 humana como isca revelou parceiros de interação diferentes daqueles reportados para Nip7p em levedura. FTSJ3 foi a parceira isolada com maior frequência. FTSJ3 é a provável ortóloga de Spb1p em levedura, a qual está envolvida na formação da subunidade ribossomal 60S. A associação entre FTSJ3 e NIP7 foi demonstrada por ensaios de pull-down e imunoprecipitação, como sendo dependente de RNA. A co-localização nucleolar e co-sedimentação dessas proteínas em fracionamento em gradiente de sacarose corroboram a associação. Além disso, células humanas deficientes em FTSJ3 revelaram defeitos na via de maturação do rRNA 18S, mesma via afetada pela depleção de NIP7. Em adição, a caracterização proteômica de complexos contendo FTSJ3 e NIP7 revelaram que essas proteínas co-purificam complexos pré-ribossomais. Uma comparação entre o conjunto de proteínas que interagem com Spb1p e as proteínas identificadas nos ensaios de pull-down com FLAG-FTSJ3 revelou que elas apresentam apenas um ortólogo em comum, o qual, incrivelmente, é Nip7/NIP7. Essas observações revelaram diferenças significativas na função desses fatores durante a síntese de ribossomos em levedura e células humanas, adicionando NIP7 e FTSJ3 na lista crescente de fatores com funções divergentes nas vias de processamento do rRNA em levedura e humanos
Abstract: Previous studies from our laboratory have demonstrated the interaction between the SBDS and NIP7 human proteins. SBDS play a role in ribosome biogenesis and its deficiency is associated to the Shwachman-Bodian-Diamond syndrome. NIP7 is a conserved protein and has already been characterized in yeast, where it participates in the 60S ribosomal subunit formation. In this work, we investigated the role of NIP7 in ribosome biogenesis in human cells. NIP7 knockdown caused pre-rRNA processing defects associated to the 18S rRNA maturation, leading to deficiency in 40S ribosomal subunit synthesis. The divergence between NIP7 function in yeast and human cells is further supported by the fact that human NIP7 does not complement yeast deficient in Nip7p. In addition, a two-hybrid screen using human NIP7 as bait revealed interaction partners different from those reported for yeast Nip7p. FTSJ3 was isolated as one of the most frequent human NIP7-interacting candidates. FTSJ3 is a putative ortholog of yeast Spb1p, which has been implicated in 60S ribosomal subunit synthesis. The association between FTSJ3 and NIP7 was showed by pull-down and immunoprecipitation assays as an RNA-dependent interaction. Nucleolar colocalization and co-sedimentation on a sucrose gradiente fractionation corroborate this association. Furthermore, RNAi-mediated knockdown revealed that depletion of FTSJ3 causes pre-rRNA processing defects in the pathway leading to 18S rRNA maturation, the same pathway affected by NIP7 downregulation. In additon, proteomic characterization of FTSJ3- and NIP7- containing complexes showed that these proteins copurify pre-ribosomal complexes. A comparison of the set of Spb1p-interacting proteins with the proteins identified in the pulldown with FLAG-FTSJ3 showed that they share only one ortholog which, incredibly, is Nip7/NIP7. These observations revealed significant differences in the function of these factors during the synthesis of ribosomes in yeast and human cells, adding NIP7 and FTSJ3 to the growing list of factors with different functions in yeast and human rRNA processing pathways
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Bendele, Kylie Gayle. "Molecular characterization of Theileria spp. using ribosomal RNA". Texas A&M University, 2004. http://hdl.handle.net/1969.1/2649.
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The molecular characterization of twenty six Theileria spp. isolates and one C. felis isolate were done on the small subunit ribosomal RNA (SSU rRNA) gene, the 5.8S gene, and the two internal transcribed spacer regions using gDNA. The SSU rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (Chae et al. 1998; Chae et al., 1999 a,b,c; Stockham et al., 2000; Cossio-Bayugar et al., 2002; Gubbels et al., 2000). The genomic region consists of the internal transcribed spacer 1 (ITS 1), the 5.8S gene, and internal transcribed spacer 2 (ITS 2) (ITS 1-5.8S-ITS 2 gene region) and separates the SSU rRNA gene from the large subunit ribosomal RNA gene. The 5.8S rRNA gene is highly conserved in size and nucleotide sequence, is relatively constant in molecular weight, and has an average chain length of approximately 160 nucleotides and has proven useful in dividing subgenera of Gyrodactylus ((Nazar, 1984; Zietara et al., 2002).
Pairwise comparisons were done between the clones of an individual isolate and among the clones of the different isolates. Phylogenetic trees were made from the resulting sequences. This study shows that different SSU rRNA genes may be associated with ITS 1-5.8S-ITS 2 gene regions of distinct sequence in the same isolate. This study also demonstrates that considerable ITS 1-5.8S-ITS 2 gene region sequence variation may exist within a species. This may be useful for subspeciation designation, or may simply reflect considerable variation within the population. This study shows that the ITS 1-5.8S-ITS 2 gene region may be a useful molecular marker for the taxonomy of Theileria spp.
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Stults, Dawn Michelle. "STRUCTURAL INSTABILITY OF HUMAN RIBOSOMAL RNA GENE CLUSTERS". UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/68.
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The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity.
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Wicks, Benjamin. "Sensitive and rapid determination of specific ribosomal RNA". Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389572.
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Qin, Daoming. "Role of 16S Ribosomal RNA in Translation Initiation". The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299007063.
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Leipuvienė, Ramunė. "Frameshifting as a tool in analysis of transfer RNA modification and translation /". Umeå : Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-302.
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TAVERNITI, VALERIO. "RNA MATURATION/DEGRADATION IN MYCOBACTERIA: IN VIVO AND IN VITRO CHARACTERIZATION OF RNASE J AND RNASE E". Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/151782.
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Escherichia coli and Bacillus subtilis have very different sets of ribonucleases, in particular the presence of RNase E and RNase J, respectively, that have been used to explain significant differences in RNA metabolism between these the two model organisms. However, these studies might have somewhat polarized our view of RNA metabolism, while recent works outline models of RNA degradation that are more similar than has been thought. In fact, the recent characterization of RNase Y as the scaffold for the degradosome assembly in B. subtilis lead to the consideration that RNA degradation in B. subtilis might begin through an endonucleolitycal cleavage, followed by exonucleolytical degradation.
In this work, we have identified a functional RNase J in Mycobacterium smegmatis and characterized its in vitro 5’-3’ exo- and endonucleolytic activities. Furthermore, we constructed two mutants in M. smegmatis rnj: a conditional and a knock out mutant, thus demonstrating that in M. smegmatis the gene is not essential, contrary to the RNase J1 function in B. subtilis.
In M. smegmatis RNase J co-exists with RNase E, a configuration that enabled us to study how these two key nucleases collaborate. A conditional mutant in the rne gene was constructed, demonstrating that this function is essential for M. smegmatis, as it is in E. coli. Moreover, a conditional mutant in Mycobacterium tuberculosis, confirmed its essentiality also in this organism.
We studied the respective roles of the M. smegmatis RNase J and RNAse E ribonucleases in the 5’ end maturation of the katG transcript, previously demonstrated to derive from an endoribonucleolytic processing. Here we find that RNase E is responsible of the specific cleavage of the 5’ katG end.
Further, we show that RNase E and RNase J are involved in the 5’ end processing of all three ribosomal RNAs. Thus the maturation pathways of rRNAs in M. smegmatis are quite different from those observed in both E. coli and B. subtilis. Studying organisms containing different combinations of key ribonucleases can thus significantly broaden our view of the strategies directing RNA metabolism used by various organisms.
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Trinquier, Aude. "Coupling between transfer RNA maturation and ribosomal RNA processing in Bacillus subtilis". Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7066.
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La synthèse des protéines cellulaires requiert à la fois des ribosomes fonctionnels et des ARN de transfert (ARNt) matures comme molécules adaptatrices. Les ribosomes sont de larges complexes ribonucléoprotéiques dont la biogenèse représente la plupart de la transcription cellulaire et consomme une majeure partie de l’énergie de la cellule. Par conséquent, la biogenèse des ribosomes fait l’objet d’une régulation importante afin d’ajuster le nombre de ribosomes aux besoins de la cellule et de dégrader efficacement les particules défectueuses qui pourraient interférer avec la traduction. Les ARNs ribosomiques (ARNr) et les ARNt sont tous deux transcrits sous formes de précurseurs et sont universellement maturés pour devenir fonctionnels pour la traduction. Ce travail de thèse a permis de mettre en évidence un couplage entre la maturation des ARNt et la biogenèse des ribosomes chez la bactérie modèle à Gram positif Bacillus subtilis. Ainsi, l’accumulation d’ARNt immatures lors d’une déplétion en enzymes de maturation, abolit spécifiquement la maturation en 3’ de l’ARNr 16S par l’endoribonucléase YqfG/YbeY, dernière étape dans la formation de la petite sous-unité ribosomique (30S). Nous avons mis en évidence que ce défaut de maturation résultait d’un défaut d’assemblage tardif du 30S coïncidant avec des changements d’expression de plusieurs facteurs d’assemblage du ribosome. Nous avons montré que cette modulation d’expression provenait d’effets transcriptionel et post-transcriptionel. De façon inédite, nos résultats indiquent que l’accumulation d’ARNt immatures est perçue par RelA (le facteur de la réponse stringente), déclenchant la production de (p)ppGpp. Nous avons observé que cette synthèse de (p)ppGpp et la baisse concomitante des niveaux de GTP cellulaire, inhibe la maturation de l’ARNr 16S en 3’, probablement via un blocage des GTPases impliquées dans l’assemblage des ribosomes. L’inhibition de la maturation de l’ARNr 16S côté 3’ est supposée conduire, par la suite, à une dégradation des particules partiellement assemblées par la RNase R. Ainsi, nos résultats supportent un modèle où RelA jouerait un rôle central ; en percevant une déficience de maturation des ARNt et en ajustant, en conséquence, la biogenèse des ribosomes via la production de (p)ppGpp. Ce mécanisme de couplage permettrait de maintenir un équilibre fonctionnel entre ARNt et ARNr, les deux composants majeurs de la machinerie de traductionCellular protein synthesis both requires functional ribosomes and mature transfer RNAs (tRNAs) as adapter molecules. The ribosomes are large essential ribonucleoprotein complexes whose biogenesis accounts for most of cellular transcription and consumes a major portion of the cell’s energy. Ribosome biogenesis is therefore tightly adjusted to the cellular needs and actively surveilled to rapidly degrade defective particles that could interfere with translation. Interestingly, tRNAs and ribosomal RNAs (rRNAs) are both transcribed from longer primary transcripts and universally require processing to become functional for translation. In this thesis, I have characterized a coupling mechanism between tRNA processing and ribosome biogenesis in the Gram-positive model organism Bacillus subtilis. Accumulation of immature tRNAs during tRNA maturase depletion, specifically abolishes 16S rRNA 3’ processing by the endonuclease YqfG/YbeY, the last step in small ribosomal subunit formation. We showed that this maturation deficiency resulted from a late small subunit (30S) assembly defect coinciding with changes in expression of several key 30S assembly cofactors, mediated by both transcriptional and post-transcriptional effects. Interestingly, our results indicate that accumulation of immature tRNAs is sensed by the stringent factor RelA and triggers (p)ppGpp production. We showed that (p)ppGpp synthesis and the accompanying decrease in GTP levels inhibits 16S rRNA 3’ processing, most likely by affecting GTPases involved in ribosome assembly. The inhibition of 16S rRNA 3’ processing is thought to further lead to degradation of partially assembled particles by RNase R. Thus, we propose a model where RelA senses temporary slow-downs in tRNA maturation and this leads to an appropriate readjustment of ribosome biogenesis. This coupling mechanism would maintain the physiological balance between tRNAs and rRNAs, the two major components of the translation machinery
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Leplus, Alexis. "Study of factors implicated in small ribosomal subunit biogenesis under differents growth conditions". Doctoral thesis, Universite Libre de Bruxelles, 2010. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210189.
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La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse :les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers :Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.Doctorat en Sciences
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Lepore, Nathalie. "La surveillance nucléolaire: étude des mécanismes de dégradation des ARN ribosomiques". Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209830.
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La biogenèse des ribosomes est un processus hautement complexe impliquant plusieurs centaines de facteurs de synthèse dont la finalité est la production de toutes les protéines de la cellule. Chaque étapes de la ribogenèse est une source potentielle d’erreur et est vérifiée par des mécanismes de contrôle de qualité redondants et rigoureux. Dans la première partie de ma thèse, j’ai collaboré à une meilleure compréhension d’une des voies de la surveillance nucléolaire, celle qui dégrade les pré-ribosomes défectueux recrutée à partir de l’extrémité 3’ des ARN ribosomiques (ARNr). Suite à une erreur d’assemblage, les pré-ARNr sont polyadénylés par le complexe nucléaire TRAMP, ce dernier est recruté cotranscriptionnellement. Les ARNr polyadénylés deviennent alors des substrats pour l’exosome et sont dégradés.
On ignore comment la synthèse des ARNr, leur maturation et la surveillance nucléolaire sont intégrées mais on suspecte l’existence d’une interface physico-fonctionnelle à l’ADNr. Dans une deuxième partie de ce travail, nous avons testé si des cofacteurs de l’exosome, les protéines Nrd1/Nab3 étaient impliquées dans la surveillance des pré-ARNr. Nous rapportons que, chez la levure S. cerevisiae, le facteur d’élongation Spt5 interagit avec l’ARN polymérase (Pol) I et avec Nrd1. L’interaction entre Spt5 et ces deux protéines requiert la présence d’un domaine particulier situé à l’extrémité C-terminal ressemblant au « Carboxy-terminal domain » (CTD) de la Pol II appelé « Carboxy terminal repeat » (CTR). Spt4/Spt5 et Nrd1/Nab3 interagissent fonctionnellement avec Rrp6, sous-unité catalytique de l’exosome. Ces complexes colocalisent à l’ADNr, comme déterminé par ChIP. Des mutations dans le domaine de liaison à l’ARN (RRM – « RNA recognition motif ») de Nrd1 mais pas dans son domaine de liaison au CTD (CID – « carboxy-terminal interacting domain ») de la Pol II et dans le RRM de Nab3 mènent à l’accumulation de transcrits ribosomiques aberrants polyadénylés. Ceci indique que Nrd1/Nab3 contribue au recrutement de la surveillance nucléolaire à la Pol en cours d’élongation pour « scruter » les transcrits ribosomiques naissants.
Nous proposons un modèle dans lequel Nrd1/Nab3 sont recrutées à la machinerie d’élongation de la transcription via leur interaction avec Spt5 afin de surveiller la synthèse des transcrits ribosomiques. Si un problème dans la fabrication des transcrits naissants a lieu, des sites de liaison pour Nrd1/Nab3 sur les pré-ARNr normalement recouverts de facteurs de synthèse seraient dénudés. Ceci marquerait les transcrits ribosomiques aberrants et entrainerait leur dégradation par les machineries de dégradation TRAMP et exosome.
Doctorat en Sciences
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Hsiao, Chiaolong. "Computational bioinformatics on three-dimensional structures of ribosomes using multiresolutional analysis". Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26634.
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Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.Committee Chair: Williams, Loren; Committee Member: Doyle, Donald; Committee Member: Harvey, Stephen; Committee Member: Hud, Nicholas; Committee Member: Wartell, Roger. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Ciriello, Giovanni. "Structural studies of the ribosome: rRNA building blocks characterization and interactions analysis". Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3426117.
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The first high resolution crystal structures initiated a new era in ribosomal studies in which investigations depend on knowledge of atomic coordinates. The studies I conducted on the three-dimensional structure of the ribosome aim to reveal and understand the ribosome modular organization, and at the same time to investigate protein-RNA interaction mechasisms. All the results presented in this thesis refer to the case study
of the large ribosomal subunit 50S of Haloarcula marismortui, omitting the 5S rRNA.
Three-dimensional RNA motifs are the basic building blocks of ribosomal RNA (rRNA) architecture. A novel method to search for these substructures, based on shape histograms, is presented in Chapter 2. The shape histogram is a vector representation of the distribution of distances between a set of points and a fixed one. Shape histograms are not only an efficient tool to compare RNA fragments, but also they well capture the structural similarities behind the flexible and highly variable structure of ribosomal RNA. The method outperforms existing ones in time efficiency, reaching the same level of correctness.
Branching points of three or more helices, named junctions, are the most variable and complex loops of rRNA, and the least characterized. Shape histograms together with angles between inter-helical fragments provide major informations on the 3D conformation of junctions: angles give informations on the eccentricity of the loop, while shape histogram range and distribution reveal the folding. Based on these features I
propose a possible classification of these motifs with respect to their 3D conformation.
Finally in Chapter 4 I dissect protein-RNA interactions within the ribosome. Statistical analysis reveal distinguishing features of the ribosome with respect to protein interactions with other RNA molecules, such as the dominant role of the ribose group.
Furthermore protein interaction mechanisms with known RNA motifs are investigated, in particular with standard tetraloops, kink-turns, and single extruded nucleotides in general. A consensus interaction pattern is detected for protein contact surfaces with standard tetraloops, characterized by dense areas of contacts made by positive residues (mainly arginine). Ribosomal proteins also reveal a characteristic binding site with
kink-turns in correspondence of the extruded base. Due to its shape, this site has been called tripod. Tripods proved to be common to several single extruded nucleotides. Besides
a conserved shape, tripods show a preference towards purines, especially adenine, and typically make hydrogen bonds.Le prime strutture cristallizate ad alta risoluzione diedero inizio ad una nuova era negli studi sui ribosomi, era nella quale la ricerca beneficia delle coordinate spaziali di ogni singolo atomo. Gli studi che ho condotto sulla struttura tridimensionale del ribosoma puntano a compredere l’organizzazione modulare della molecola, ed allo stesso tempo ad investigare i meccanismi di interazione proteine-RNA. Tutti i risultati presentati in questa tesi riferiscono alla subunità principale 50S di Haloarcula marismortui, omettendo il filamento di RNA 5S. Motivi tridimensionali ricorrenti sono i mattoni basilari dell’architettura dello RNA ribosomiale: essi sono caratterizzati da una struttura 3D conservata, e ricorrono frequentemente all’interno della molecola. Un nuovo metodo per ricercare queste sotto strutture, basato su shape histogram, è presentato al Capitolo 2. Lo shape histogram è una rappresentazione vettoriale della distribuzione delle distanze di un insieme di punti da uno fissato. Gli shape histogram non sono solo strumenti efficienti per confrontare frammenti diversi di RNA, sono inoltre in grado di catturare le similarità strutturali nascoste dalla struttura flessibile ed altamente variabile dello RNA ribosomiale. Il metodo infatti si dimostra più efficiente di quelli noti in letteratura, ed ugualmente efficace in termini di correttezza dei risultati. Tra i loop studiati, le giunzioni di tre o più eliche sono il tipo più variabile e complesso, ed il meno caratterizzato. Gli shape histogram insieme alle sequenze di angoli formati dai frammenti congiungenti più eliche sono in grado di dare molte informazioni sulla conformazione tridimensionale di queste giunzioni: gli angoli forniscono indicazioni sull’eccentricità di una giunzione, mentre range e distribuzione dei valori degli shape histogram rivelano il folding. Basandosi su queste caratteristiche, propongo una classificazione per questi motivi rispetto alla loro conformazione nello spazio. Infine il Capitolo 4 esamina le interazioni proteine-RNA all’interno del ribosoma. Osservazioni di tipo statistico rivelano caratteristiche distintive del ribosoma rispetto alle interazioni proteiche con altri tipi di RNA, come ad esempio il ruolo dominante del gruppo ribosio. Inoltre vengono studiati i meccanismi di interazione delle proteine con i motivi strutturali, in particolare standard tetraloop, kink-turn e basi esposte. Un pattern comune di interazioni è rintracciato per le superfici di contatto formate dalle proteine con i tetraloop, caratterizzato da zone dense di interazioni fatte da aminoacidi a carica positiva (principalmente arginine). Le proteine ribosomiali rivelano inoltre un sito di contatto caratteristico nelle interazioni con i kink-turn, in corrispondenza della base esposta. Per la sua forma questo sito è stato chiamato tripod, ovvero tripode. I tripodi si sono dimostrati essere in realtà comuni a molti nucleotidi non accoppiati la cui base è esposta. Oltre ad una conformazione tridimensionale conservata, i tripodi dimostrano una preferenza verso le purine, soprattutto adenina, e tipicamente formano legami idrogeno.
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Perez, Fernandez Déborah. "Aminoglycoside antibiotics to selectively target bacterial 16S ribosomal RNA /". Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17284.
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Lévesque, Madeleine. "Investigation of alder (Alnus incana) chloroplast ribosomal RNA genes". Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5571.
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In an effort to position the alder, Alnus incana, in the scope of phylogenetic relationships, ribosomal RNA genes from its chloroplasts have been cloned and analysed. A genomic library was constructed in the vector LambdaGEM-11 with DNA isolated from leaf tissue. DNA fragments covering the length of the 23S and 16S rRNA genes as well as trnI and sections of trnA were sequenced. The order of chloroplast genes within the sequenced region of alder is identical to that seen in higher plants: 16S-trnI-trnA-23S. Comparisons of the primary sequence of the alder 16S and 23S rRNA genes with available ribosomal RNA and DNA sequences have demonstrated the greatest degree of homology between the alder sequences and those of other higher plant sequences. Comparisons to bacterial sequences yield higher homologies to the cyanobacterial sequences as opposed to those of E. coli. Further sequence analysis shows that the primary sequence obtained for the 16S rRNA gene in alder can be folded to follow the secondary structure of the maize 16S rRNA molecule. (Abstract shortened by UMI.)
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Wiener, Christopher Charles. "Intraspecific 16S ribosomal RNA gene polymorphism in Staphylococcus epidermidis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ39895.pdf.
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Baylis, H. A. "The ribosomal RNA genes of Streptomyces coelicolor A3(2)". Thesis, University of East Anglia, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374251.
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Firek, Simon. "The promotion of ribosomal RNA transcription in Xenopus laevis". Thesis, University of Portsmouth, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236392.
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Russell, Ian Douglas. "NOP3, a protein involved in pre-ribosomal RNA processing". Thesis, Open University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239708.
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Zhang, Ze. "The control of ribosomal RNA synthesis in mammalian cells". Thesis, University of Southampton, 2013. https://eprints.soton.ac.uk/350477/.
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The biogenesis of ribosomes is a fundamental process that occurs in all living cells. In mammalian cells, it is a highly complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNAs) with about 80 ribosomal proteins (RPs). More than 150 non-ribosomal proteins are involved in the processing of rRNAs. The main focus of this project is to use adult rat ventricular cardiomyocytes (ARVCs) as a model to address how mTOR complex 1 (mTORC1) and other signalling pathways regulate the synthesis of rRNAs. A new technique has been developed to monitor the synthesis of new rRNAs using 4-thiouridine (4-SU) and I have applied it in both HeLa cells and heart muscle cells to study the control of ribosome synthesis. HeLa cells were treated with different mTOR inhibitors to identify effects on the transcription and/or decay of rRNA. We analysed both the synthesis rate and the decay rate of new RNAs made by Pol I and Pol III using real-time RT-PCR. Interestingly, rapamycin not only blocked the synthesis of 18S, 28S and 5S rRNA, but also induced the decay of newly synthesized rRNAs. This demonstrates that mTORC1 regulates Pol I and Pol III transcription, as well as the decay of rRNA. In cardiomyocytes, hypertrophic agents such as phenylephrine (PE) strongly activate protein synthesis and lead to heart cell growth. The boost of protein synthesis drives the increase of cell size and leads to hypertrophy. Cardiac hypertrophy (CH) is a major risk factor for heart failure. Therefore, it is important to understand the mechanisms that how hypertrophic agents which cause the overgrowth of heart muscle increase ribosome production. Although it is known that inhibiting mTORC1 largely blocks the rapid activation of protein synthesis by PE, here it did not affect the synthesis of new 18S rRNAs. However, inhibitors of the MEK/Erk pathway and p90RSK each block the new rRNA synthesis. These data reveal that, in contrast to many other types of cell, ribosome biogenesis is controlled by MEK/ERK/p90RSK signalling, not mTORC1, in cardiomyocytes. Taken together, the data presented here may provide cues for potential valuable therapy of cardiac left ventricular hypertrophy.
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Dove, Chris Hays. "Models for the formation of 5.8S ribosomal RNA dimer". Thesis, Virginia Polytechnic Institute and State University, 1985. http://hdl.handle.net/10919/50088.
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Ribosomal 5.85 RNA is a component of the large (605) ribosomal subunit in eucaryotes. Studies of 5.85 rRNA in solution have shown that under certain conditions, including standard isolation procedures, the molecule complexes with itself to form dimers and higher multimers. Two models have been proposed in the literature to explain the intermolecular interactions responsible for 5.85 rRNA dimer formation. The terminal interaction model of Sitz et al. (Biochem. 17, 5811-5815, 1978) proposes that the dimer forms through base-pairing of the 5' and 3' terminal sequences of two 5.85 rRNA molecules. Pavlakis et al. (Nucl. Acids Res. 7, 2213-2237, 1978) showed that 5.85 rRNA lacking the 3' terminal region was capable of forming dimer. They proposed an alternative model for 5.85 rRNA dimer formation in which an entirely different part of the molecule interacts to form a double-stranded palindrome.
In this study, enzymatic probing techniques and stability measurements (both experimental and theoretical) were used to determine which model most accurately describes the intermolecular interactions of the 5.88 rRNA dimer. The methods used for determining stability were not able to discriminate between the models for 5.88 rRNA dimer formation. Results from the structural probing studies, however, support the terminal interaction model and indicate that the palindrome interaction does not occur in 5.88 rRNA dimer formed from the intact molecule.Master of Science
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Herdman, Chelsea. "Relative roles of UBF and RRN3 in the transcription of the ribosomal RNA genes and ribosome biogenesis determined using in vivo mouse models". Doctoral thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/28387.
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La biogenèse des ribosomes, aussi appelée la synthèse ribosomale, est un processus cellulaire important se déroulant dans le nucléole et implique la transcription par les trois ARN polymérases nucléaires. L’étape initiale et limitante de ce processus est la transcription des ARNs ribosomaux catalytiques, 28S, 18S and 5.8S, sous la forme d’un long précurseur d’ARN ribosomal (pre-ARNr/47S) par l’ARN polymérase I (RPI). RPI possède un ensemble de facteurs de transcription généraux responsables de son activation. Ces facteurs sont la protéine architecturale UBF, le facteur SL1 qui contient TBP, le facteur d’initiation RRN3 et le facteur de terminaison TTF1. La synthèse de l’ARN ribosomale est finement régulée et correspond à 30-50% de l’ensemble de la transcription de la cellule. De plus, ce processus est lié à la croissance cellulaire, la transformation, la prolifération et à l’activité des facteurs suppresseurs de tumeurs et des oncogènes. UBF et RRN3 sont notamment activés par plusieurs voies de signalisation de croissance cellulaire. Dans les cellules de mammifère, il existe ~200 copies d’ADNr par génome haploïde. Les fragments répétés d’ADN ribosomal sont arrangés en répétition en tandem sur les bras courts des chromosomes acrocentriques. De façon intéressante, dans les cellules somatiques, seulement la moitié des copies d’ADNr sont actives, alors que les autres sont maintenues dans une forme inactive par les modifications épigénétiques et la formation d’hétérochromatine. La raison pour laquelle le génome contient autant de copies et la régulation de leur activité ne sont pas bien comprises. Cette thèse présente l’analyse de l’importance in vivo d’UBF et de RRN3 pour la régulation de la transcription de l’ARNr et pour le maintien de la structure chromatinienne de l’ADNr. Nous avons précédemment analysé la perte de fonction de UBF dans les fibroblastes embryonnaires de souris en utilisant le système de perte de fonction conditionnelle dépendante du tamoxifène. Puisque l’un de nos objectifs était de comparer la fonction de RRN3 dans un modèle similaire, nous avons réanalysé la perte de fonction de RRN3 chez la souris et généré des lignées cellulaires comme préalablement réalisées avec la perte de fonction d’UBF. Nous avons déterminé que RRN3 est essentiel à la préimplantation et le développement est arrêté à E3.5, ce qui contredit les résultats obtenus par un autre groupe qui avait obtenu un arrêt du développement beaucoup plus tardif, à E9.5. Une lignée de fibroblastes embryonnaires de souris inductible au tamoxifène a été créée pour RRN3 de façon similaire à ce qui avait été fait pour UBF. La perte de fonction d’UBF ou de RRN3 inhibe la transcription par RPI. Par contre, nous démontrons que UBF est responsable du recrutement à l’ADNr des autres facteurs associés à RPI et du maintient de l’état ouvert de la chromatine. En comparaison, RRN3 est requis simplement pour le recrutement de RPI. Dans cette étude, nous avons également identifié une région frontalière en amont de l’ADNr formée de H2A.Z, TTF1, CTCF et des modifications d’histones activatrices. Nous avons également découvert que la perte d’UBF entraine une mort cellulaire synchronisée par apoptose, indépendamment de p53 et ce spécifiquement dans les lignées cellulaires transformées. Ce résultat suggère qu’il pourrait être possible de cibler UBF dans le traitement contre le cancer puisque la perte de UBF dans les lignées cellulaires primaires cause un arrêt de prolifération sans entrainer l’apoptose. Finalement, nous avons observé que le niveau d’activité de l’ADNr dans les cellules pluripotentes est différent que dans les cellules différenciées. Des lignées de cellules souches embryonnaires (ESCs) ont été générées à partir des souris conditionnelles pour UBF et RRN3 et nos résultats préliminaires suggèrent que la totalité des gènes de l’ADNr est active dans les cellules pluripotentes. Ce modèle est idéal pour étudier la régulation de l’ADNr ainsi que le rôle de UBF et RRN3 dans cette régulation après l’induction de la différentiation. En résumé, ces résultats permettront de clarifier le rôle in vivo de UBF et RRN3 dans la transcription de l’ARN ribosomal et dans le maintien de l’intégrité de l’ADNr.Ribosome biogenesis, or the synthesis of ribosomes, is an important cell process occurring in the nucleolus that utilizes transcription by all three nuclear RNA polymerases. The initial and rate-limiting step is the transcription of the catalytic ribosomal RNAs 28S, 18S and 5.8S in the form of a precursor ribosomal RNA (pre-rRNA/47S) by RNA polymerase I (RPI, also known as Pol1 and POLR1). RPI has a dedicated set of basal factors responsible for its activation. These are the architectural factor UBF, the TBP containing factor SL1, the initiation factor RRN3, and the termination factor TTF1. Ribosomal RNA synthesis is tightly regulated and accounts for 30-50% of total gene transcription. As such, this process is linked to cell growth, transformation, proliferation and the actions of tumour suppressors and oncogenes. Notably, UBF and RRN3 are activated by many of the same growth signaling pathways. The human and mouse haploid genome contain ~200 copies of the ribosomal RNA genes, the ribosomal DNA (rDNA). These ribosomal DNA copies are arranged in tandem repeats on the short arms of acrocentric chromosomes. Interestingly, only a fraction of the rDNA copies are active, and a significant number are epigenetically silenced and heterochromatic. The reason for having so many copies and their regulation in vivo by silencing is not yet understood, though it has been connected with genome stability. This thesis presents the analysis of the in vivo requirements for UBF and RRN3 in rRNA transcription and rDNA chromatin structure. We had previously analyzed the loss of UBF in mouse embryonic fibroblasts using tamoxifen-dependent conditional knockout. As we wanted to compare the loss of RRN3 in a similar model, we re-analyzed the RRN3 knockout mice and created cell lines as was performed for the UBF knockout. Importantly, we find that RRN3 is essential for preimplantation and its loss arrests development at E3.5, contrary to previous work that showed a late E9.5 developmental arrest. Using mouse embryonic fibroblast (MEF) cell lines conditional for UBF or RRN3, we found that the loss of either factor prevented RPI transcription. However, we found that UBF was essential for the recruitment of the other RPI transcription factors and the formation of the preinitiation complex, as well as to maintain an open rDNA chromatin structure, while RRN3 was required only for RPI recruitment. These studies allowed us to identify an upstream boundary element on the rDNA formed of H2A.Z, TTF1, CTCF and activating histone marks, which is independent of RPI activity. We also found that UBF loss, but not RRN3 loss, led to a synchronous and massive p53-independent apoptosis, specifically in oncogenically transformed cells. This strongly suggests that drug targeting UBF could be a viable cancer treatment. Finally, we have observed that the rDNA activity status in pluripotent cells differs from that of differentiated cells. Embryonic stem cells (ESCs) were also generated from the mice conditional for UBF and RRN3. Preliminary results indicate that, unlike somatic cells, all the rRNA genes in these and other pluripotent cell lines are potentially active. This makes ESCs and their differentiation an ideal model in which to study the establishment of rDNA silencing and the role of UBF and/or RRN3 in this process. Together these data define the in vivo roles of UBF and RRN3 in ribosomal RNA transcription and suggest mechanisms by which they maintain rDNA integrity and may drive cell differentiation.
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Webb, Vera Ann B. "In vivo in vitro synthesis of ribosomal RNA in bacillus subtilis". Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29448.
Texto completoResumen
The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes.
The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription
activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Soop, Teresa. "Assembly and transport of messenger and ribosomal RNP particles in the dipteran Chironomus tentans /". Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-521-2/.
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Stults, Dawn Michelle. "Human ribosomal RNA gene clusters are recombinational hotspots in cancer". Lexington, Ky. : [University of Kentucky Libraries], 2009. http://hdl.handle.net/10225/1122.
Texto completoResumen
Thesis (M.S.)--University of Kentucky, 2009.Title from document title page (viewed on May 6, 2009). Document formatted into pages; contains: v, 27 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 25-26).
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Tam, Man-wah. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing". Click to view the E-thesis via HKUTO, 2002. http://sunzi.lib.hku.hk/hkuto/record/B31970783.
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Li, Kwan-hing. "Identification of bacterial pathogens by 16S ribosomal RNA gene sequencing". Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31971982.
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