As the gateway to translation, ribosome biogenesis is a core cellular process that is highly efficient, accurate and regulated. This is made possible in part by a suite of ancillary proteins with diverse but poorly understood functions. One such factor, the Escherichia coli GTPase YjeQ, is suspected of playing a critical role in the assembly of the 30S ribosomal subunit. Here we demonstrate that the absence of this factor in vivo leads to an accumulation of a late-stage immature 30S subunit species. While these precursors lack several ribosomal proteins and feature a number of conformational abnormalities, they are competent for maturation, suggesting that they represent an assembly intermediate. We further demonstrate that YjeQ accelerates the maturation of these precursors in vivo. In addition, we explore the role of YjeQ through genetic interaction studies and substantiate a functional connection with the putative assembly factor RbfA. A linear correlation between growth rate and ribosomal content has been observed for multiple wild-type microbes. We have examined this relationship in the ΔyjeQ strain and found there to be a significant increase in the total cellular ribosomal material in comparison to the wild-type. This phenotype is not wholly exclusive to perturbations in biogenesis. Indeed, linear correlations and elevated levels of ribosomal content are also observed for several translation mutants. The degree of elevation, however, is marginal in comparison to that seen in the biogenesis mutant. Our work explores this phenomenon and the possibility of exploiting it to identify and further characterize perturbations in the ribosome assembly process. / Thesis / Doctor of Philosophy (PhD) / In all cells, translation is carried out by ribosomes, large molecules that mediate the interpretation of the genetic code. These cellular interpreters are absolutely required for protein synthesis in bacteria and thus, are necessary for life. Like proteins, ribosomes themselves must also be synthesized, a process known as ribosome biogenesis. The ribosome consists of myriad RNA and protein components and is perhaps the single most complex machine in cells. Nevertheless, cells can build these enormous molecules in less than two minutes. This is made possible by a team of helper proteins, such as the bacterial assembly factor YjeQ. The function of this protein has evaded researchers, but there is growing evidence that it facilitates a key stage in the assembly process. Our work provides new detail into how this protein influences ribosome biogenesis, and how this in turn affects the overall health and proliferation of bacterial cells.

Antibiotic resistance in bacteria is becoming a major threat to public health. Many of the antibiotics used today in the clinic target the process of protein synthesis performed by the ribosome. Recent prospects for blocking ribosome function are increasingly focusing on preventing the assembly of bacterial ribosomes. A number of ribosome assembly factors are emerging as attractive targets for novel antibiotics that work in new ways. YphC and YsxC are essential GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit; however, their roles in this process are still uncharacterized. To explore their function, we biochemically and structurally characterized the 45SYphC and 44.5SYsxC precursor particles accumulated from strains depleted of YphC and YsxC, respectively. Quantitative mass spectrometry analysis and 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in maturation of functional sites of the 50S subunit. We also observed that YphC and YsxC bind specifically to the two immature particles. In addition, we characterized the structure of the 50S subunits in complex with the RbgA protein. The preliminary 3D structure shows that the RbgA protein binds to the P site of the 50S subunit and displaces h69. There are also missing densities in the structure for h68 and the uL16 ribosomal protein. We expect that the atomic resolution structure of the 50S.RbgA complex will reveal the function and molecular mechanisms of this assembly factor. The deep structural understanding of protein synthesis process done by the ribosome led to the optimization of over a hundred antibiotics that are currently used in thev clinic. In the same manner, work described in this thesis provides novel insights into understanding the maturation of the large ribosomal subunit, and is paving the way to use the bacterial ribosome biogenesis pathway as a target for the development of new antimicrobials. / Thesis / Doctor of Philosophy (PhD)

No description available.

Ribosome biosynthesis in eukaryotes is a complex process involving over 200 accessory factors. Nob1, Dim2, and Tsr1 are three conserved factors that are all involved in the late processing steps of the small subunit (40S) pre-rRNA. Depletion of any of these factors leads to the accumulation of the immature 20S pre-rRNA. Nob1, an essential protein in yeast, performs the final cleavage of small subunit rRNA giving rise to the mature particle. It is aided in this process by other proteins such as Dim2. Previously, the two proteins have been shown to interact. Nob1 function was found to be more efficient in the presence of Dim2. Previous studies also indicated that Nob1 binds a site on the pre-40S that is distal to the cleavage site while Dim2 binds proximally. Using analytical gel filtration, electrophoretic mobility shift assays, and isothermal titration calorimetry we show that Nob1 does not interact with the distal binding site in vitro. Instead, a stable complex with a micromolar disassociation constant can be formed with a sequence derived from the cleavage site. Thus, Nob1 and Dim2 appear to be competing for this site. The interaction with both proteins is blocked when this sequence is sequestered in a hairpin structure, which has been previously predicted to form at this site. By altering individual bases in the RNA sequence, we have identified the sequence determinants for Nob1-rRNA recognition. Tsr1 function is unknown to date. It shares sequence similarity with certain GTPases; however, no GTP binding has been identified in previous studies. The depletion of this factor leads to a similar phenotype as the depletion of Nob1 and Dim2. By screening various deletion constructs, we have obtained good quality, diffracting crystals of yeast Tsr1. However, due to time constraints, the full structure has not been solved. Here we present the initial analysis of the crystallographic data and the potential for solving the structure in the future. Overall, the data presented in this thesis bring insight into the final step of small subunit ribosome maturation.

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.

Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

No description available.

The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)

A biogênese do ribossomo é um processo complexo, altamente ordenado e regulado, no qual o transcrito primário é processado por endo e exonucleases para gerar os RNAs ribossomais maduros. Este processo foi melhor caracterizado em Saccharomyces cerevisiae, porém alguns fatores atuantes em humanos tiveram uma função divergente descrita. Um desses fatores é a proteína NIP7, altamente conservada em eucariotos, que atua na formação da subunidade ribossomal 60S, em levedura, e 40S, em humanos. Assim, esse trabalho propôs a caracterização funcional da proteína CrNIP7, homóloga a NIP7, presente em Chlamydomonas reinhardtii. C. reinhardtii é uma alga verde unicelular ancestral a plantas, utilizada como modelo eucarioto para estudos de fotossíntese e de flagelos. Nesse trabalho, um estudo de complementação funcional foi realizado utilizando duas linhagens de Saccharomyces cerevisiae diferentes e em ambas CrNIP7 complementou a função de Nip7p de leveduras, indicando uma participação na síntese da subunidade 60S do ribossomo. Uma busca por parceiros de interação de CrNIP7 foi também realizada, utilizando CrNIP7 como isca para rastrear uma biblioteca de cDNA de C. reinhardtii em sistema de duplo híbrido em leveduras, o que resultou em dois novos potenciais parceiros de interação. Esses parceiros foram identificados como proteínas preditas conceitualmente no genoma de C. reinhardtii, denominadas Predicted e G-patch. Adicionalmente, a interação entre CrNIP7 e CrSBDS, proteína homóloga a Sdo1 (de levedura) e HsSBDS (de humanos), foi confirmada através de um experimento de duplo híbrido dirigido. A interação entre as proteínas CrNIP7 e CrSBDS foi validada por pull down e um teste preliminar sugeriu que CrNIP7 e Predicted também interagem in vitro. Análises de bioinformática indicam que Predicted, G-patch e CrSBDS tenham regiões intrinsicamente desordenadas, as quais podem se estruturar na interação com seus parceiros. Em conjunto, os resultados desse trabalho contribuem para entendimento do papel de CrNIP7 na biogênese de ribossomos em Chlamydomonas reinhardtii em comparação com outros modelos eucarióticos. / Ribosome biogenesis is a complex, highly regulated and ordered process in which the primary transcript is processed by endo- and exonucleases to generate the mature ribosomal RNAs. This process was best characterized in Saccharomyces cerevisiae, but some factors have been described in humans with different function. One of these divergent factors is NIP7, a highly conserved protein in eukaryotes, which acts in the formation of ribosomal 60S subunit, in yeast, and 40S, in humans. Based on this, this work proposed the functional characterization of CrNIP7 protein, homologous to NIP7, from Chlamydomonas reinhardtii. C. reinhardtii is a green alga, ancestral to plants, that is used as an eukaryote model for photosynthesis and flagella studies. In this study, a functional complementation assay was performed using two different strains of Saccharomyces cerevisiae and, in both approaches, CrNIP7 protein complemented the function of Nip7p from yeast, indicating its participation in the synthesis of the 60S ribosomal subunit. A two-hybrid assay was carried out using CrNIP7 as bait to screen a C. reinhardtii cDNA library in order to find out CrNIP7 interaction partners, wich resulted in two novel potentially partners. The interacting proteins were identified as conceptually predicted proteins in the genome of C. reinhardtii and were called Predicted and G-patch. Additionally, the interaction between CrNIP7 and CrSBDS, a protein homologous to Sdo1 (yeast) and HsSBDS (humans), was confirmed by a direct two-hybrid assay. The interaction between CrNIP7 and CrSBDS proteins was validated by pull down and a preliminary test suggested that CrNIP7 and Predicted also interact in vitro. Bioinformatics analyzes indicate that Predicted, G-patch and CrSBDS have intrinsically disordered regions, which can be ordered in the moment of interaction. Taken together, the results of this work contribute to understand the role played by CrNIP7 in ribosome biogenesis in Chlamydomonas reinhardtii compared to other eukaryotic models.

The RNA helicase Prp43p is one of the few members of the DEAH-box helicase family that is known to operate in more than one cellular process in Saccharomyces cerevisiae. With roles in ribosome biogenesis and pre-mRNA splicing, Prp43p may be important in maintaining a communication conduit between these two pathways. Our studies provide insights into how Prp43p function is regulated through the use of three cofactors, Ntr1p, Pfa1p, and Gno1p, all of which interact with Prp43p at different steps of pre-mRNA splicing or ribosome biogenesis. Each cofactor contains a unique G-patch domain and our data show that they associate with Prp43p in a mutually exclusive manner. A strong growth defect and RNA processing phenotypes are seen upon overexpression of Pfa1p due to the dominance of Pfa1p interaction with Prp43p. Moreover, excess Pfa1p precludes Prp43p from interacting with either 35S pre-rRNA or U6 snRNA, indicating this one cofactor can negatively regulate Prp43p recruitment into ribosome biogenesis and pre-mRNA splicing pathways, respectively. We have determined that Ntr1p and Gno1p are able to compete with one another for Prp43p occupancy. Similar to Ntr1p, we show that the G-patch domain of Gno1p contributes to its association with Prp43p. To further understand pathway specificity of Prp43p, we characterized conditional prp43 alleles with mutations C-terminal to the conserved RecA domains of Prp43p. These novel alleles affect pre-mRNA splicing and ribosome biogenesis, though none are mutually exclusive. Multiple prp43 alleles are deficient in tri-snRNP formation, a previously uncharacterized phenotype in prp43 mutants. The majority of our prp43 mutants display varying rRNA defects, with some alleles impacting ribosome biogenesis more severely or moderately than known prp43 ATPase mutants. To correlate the processing defects seen in each allele, we have determined the extent of association of the mutants with each G-patch protein. Altogether, our data support a working model for Prp43p in which its substrate specificity, activation, and cellular distribution is coordinated through the efforts of the three G-patch proteins in yeast and sheds light on potential mechanisms of general DExH/D helicase function and regulation. / text