No description available.

No description available.

No description available.

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)

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

In all life ribosomes are the ribonucloprotein machines in charge of decoding the genetic code and synthesizing proteins. In eukaryotes, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where the final maturation steps occur prior to their partaking in translation. My dissertation work focused on aspects of the last two known steps of the pre-60S subunit cytoplasmic maturation. In the penultimate step, the anti-association factor Tif6 is released from 60S by the concerted action of the translocase-like GTPase Efl1 and Sdo1. The release of Tif6 is necessary for the ultimate maturation step, which involves release of the export adaptor Nmd3 by the ribosomal protein Rpl10 and the putative GTPase Lsg1. Nmd3 is an essential export adaptor of the 60S subunit. Nmd3 binds to the ribosome in the nucleolus and is the last known trans-acting factor to be released from the subunit in the cytoplasm. In order to gain a better understanding of the molecular events leading to the release of Nmd3 from the 60S subunit I set out to identify the binding site of Nmd3 on 60S. In a collaboration with Dr Joachim Frank’s laboratory, we obtained a cryo-EM model of Nmd3 in a complex with 60S showing Nmd3 binding to the subunit joining face of the ribosome. This work provided the first visualization of an export factor on a ribosomal subunit. The release of the anti-association factor Tif6 requires the translocase-like GTPase Efl1. Mutations in a loop of Rpl10 which embraces the P site tRNA trapped Tif6 on the subunit. These Rpl10 mutants could be suppressed by Tif6 mutants which have weakened affinity for the subunit. Mutations in Efl1 which suppress these Rpl10 mutants were also obtained. These suppressing mutations in Efl1 mapped to regions on the translocases eEF2 and EF-G important for conformational changes during translation. These results highlight molecular signaling between the P site, involving a loop of Rpl10, and Tif6, 90Å away. I propose that Efl1 promotes a translocation-like event during biogenesis of the 60S subunit prior to its first round of bona fide translation. / text

No description available.

The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and retains a distinctive association with the microtubule cytoskeleton, comprises an elliptical patch of rhodopsin photoreceptors in the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast and serves as a model for understanding how organelles are formed and placed asymmetrically in the cell. This study describes the roles of several factors in the assembly and positioning of the eyespot. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. These proteins play interdependent roles: EYE2 and the ChR1 photoreceptor co-position in the absence of pigment granules, and the pigment granules are required to maintain the shape and integrity of the EYE2/ChR1 patch. The miniature-eyespot locus MIN2 affects eyespot size and likely regulates the amount of material available for eyespot assembly. The MLT2 locus regulates eyespot size, number, and asymmetry. A novel locus, PEY1, modulates the position of the eyespot on the anterior-posterior axis by affecting microtubule rootlet length. A working model is developed wherein rootlet microtubule-directed photoreceptor localization establishes connections in the chloroplast envelope with EYE2, which directs the site for pigment granule array assembly, and MLT2 is proposed to negatively regulate the levels of eyespot proteins.

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.