Добірка наукової літератури з теми "40S subunit"
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Статті в журналах з теми "40S subunit"
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Landry-Voyer, Anne-Marie, Sarah Bilodeau, Danny Bergeron, Kiersten L. Dionne, Sarah A. Port, Caroline Rouleau, François-Michel Boisvert, Ralph H. Kehlenbach, and François Bachand. "Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors." Molecular and Cellular Biology 36, no. 24 (October 3, 2016): 3019–32. http://dx.doi.org/10.1128/mcb.00303-16.
Повний текст джерелаАнотація:
Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3′-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L -null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.
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Zeman, Jakub, Yuzuru Itoh, Zdeněk Kukačka, Michal Rosůlek, Daniel Kavan, Tomáš Kouba, Myrte E. Jansen, Mahabub P. Mohammad, Petr Novák, and Leoš S. Valášek. "Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes." Nucleic Acids Research 47, no. 15 (July 10, 2019): 8282–300. http://dx.doi.org/10.1093/nar/gkz570.
Повний текст джерелаАнотація:
Abstract eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.
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Fringer, Jeanne M., Michael G. Acker, Christie A. Fekete, Jon R. Lorsch, and Thomas E. Dever. "Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining." Molecular and Cellular Biology 27, no. 6 (January 22, 2007): 2384–97. http://dx.doi.org/10.1128/mcb.02254-06.
Повний текст джерелаАнотація:
ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.
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Eisinger, D. P., F. A. Dick, and B. L. Trumpower. "Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits." Molecular and Cellular Biology 17, no. 9 (September 1997): 5136–45. http://dx.doi.org/10.1128/mcb.17.9.5136.
Повний текст джерелаАнотація:
QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.
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Si, Kausik, and Umadas Maitra. "The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor." Molecular and Cellular Biology 19, no. 2 (February 1, 1999): 1416–26. http://dx.doi.org/10.1128/mcb.19.2.1416.
Повний текст джерелаАнотація:
ABSTRACT Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M r 25,550), designated TIF6, has been cloned and expressed inEscherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.
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Moraleva, Anastasia A., Alexander S. Deryabin, Yury P. Rubtsov, Maria P. Rubtsova, and Olga A. Dontsova. "Eukaryotic Ribosome Biogenesis: The 40S Subunit." Acta Naturae 14, no. 1 (May 10, 2022): 14–30. http://dx.doi.org/10.32607/actanaturae.11540.
Повний текст джерелаАнотація:
The formation of eukaryotic ribosomes is a sequential process of ribosomal precursors maturation in the nucleolus, nucleoplasm, and cytoplasm. Hundreds of ribosomal biogenesis factors ensure the accurate processing and formation of the ribosomal RNAs tertiary structure, and they interact with ribosomal proteins. Most of what we know about the ribosome assembly has been derived from yeast cell studies, and the mechanisms of ribosome biogenesis in eukaryotes are considered quite conservative. Although the main stages of ribosome biogenesis are similar across different groups of eukaryotes, this process in humans is much more complicated owing to the larger size of the ribosomes and pre-ribosomes and the emergence of regulatory pathways that affect their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. This review addresses the key aspects of yeast and human ribosome biogenesis, using the 40S subunit as an example. The mechanisms underlying these differences are still not well understood, because, unlike yeast, there are no effective methods for characterizing pre-ribosomal complexes in humans. Understanding the mechanisms of human ribosome assembly would have an incidence on a growing number of genetic diseases (ribosomopathies) caused by mutations in the genes encoding ribosomal proteins and ribosome biogenesis factors. In addition, there is evidence that ribosome assembly is regulated by oncogenic signaling pathways, and that defects in the ribosome biogenesis are linked to the activation of tumor suppressors.
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Gregory, Brian, Nusrat Rahman, Ananth Bommakanti, Md Shamsuzzaman, Mamata Thapa, Alana Lescure, Janice M. Zengel, and Lasse Lindahl. "The small and large ribosomal subunits depend on each other for stability and accumulation." Life Science Alliance 2, no. 2 (March 5, 2019): e201800150. http://dx.doi.org/10.26508/lsa.201800150.
Повний текст джерелаАнотація:
The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.
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Bachand, François, Daniel H. Lackner, Jürg Bähler, and Pamela A. Silver. "Autoregulation of Ribosome Biosynthesis by a Translational Response in Fission Yeast." Molecular and Cellular Biology 26, no. 5 (March 1, 2006): 1731–42. http://dx.doi.org/10.1128/mcb.26.5.1731-1742.2006.
Повний текст джерелаАнотація:
ABSTRACT Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3Δ mutants. Importantly, nearly all of the 40S ribosomal protein-encoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.
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Bhattacharya, Arpita, Kerri B. McIntosh, Ian M. Willis, and Jonathan R. Warner. "Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis." Molecular and Cellular Biology 30, no. 23 (September 27, 2010): 5562–71. http://dx.doi.org/10.1128/mcb.00618-10.
Повний текст джерелаАнотація:
ABSTRACT A set of genome-wide screens for proteins whose absence exacerbates growth defects due to pseudo-haploinsufficiency of ribosomal proteins in Saccharomyces cerevisiae identified Dom34 as being particularly important for cell growth when there is a deficit of 40S ribosomal subunits. In contrast, strains with a deficit of 60S ribosomal proteins were largely insensitive to the loss of Dom34. The slow growth of cells lacking Dom34 and haploinsufficient for a protein of the 40S subunit is caused by a severe shortage of 40S subunits available for translation initiation due to a combination of three effects: (i) the natural deficiency of 40S subunits due to defective synthesis, (ii) the sequestration of 40S subunits due to the large accumulation of free 60S subunits, and (iii) the accumulation of ribosomes “stuck” in a distinct 80S form, insensitive to the Mg2+ concentration, and at least temporarily unavailable for further translation. Our data suggest that these stuck ribosomes have neither mRNA nor tRNA. We postulate, based on our results and on previously published work, that the stuck ribosomes arise because of the lack of Dom34, which normally resolves a ribosome stalled due to insufficient tRNAs, to structural problems with its mRNA, or to a defect in the ribosome itself.
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Kozak, M. "Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs." Molecular and Cellular Biology 9, no. 11 (November 1989): 5134–42. http://dx.doi.org/10.1128/mcb.9.11.5134.
Повний текст джерелаАнотація:
This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.
Дисертації з теми "40S subunit"
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Cerezo, Emilie. "Contribution de la signalisation RSK à la synthèse de la petite sous-unité ribosomique humaine." Thesis, Toulouse 3, 2021. http://www.theses.fr/2021TOU30288.
Повний текст джерелаАнотація:
La biogenèse des ribosomes assure l'approvisionnement en ribosomes dans la cellule pour subvenir aux besoins en synthèse protéique. La production des ribosomes est un processus très couteux en énergie. Elle mobilise les trois ARN polymérases (Pol), la machinerie de traduction, le transport nucléo-cytoplasmique, ainsi qu'une succession complexe d'étapes de maturations qui implique plus de 200 facteurs d'assemblage et de maturation (AMF). Dans les cellules de mammifères en prolifération, le taux de synthèse des ribosomes a été estimé à 7500 sous-unités ribosomiques par minute, ce qui nécessiterait l'apport de ~300 000 protéines ribosomiques (RP), 150 petits ARN nucléolaires (snoARN) par ARN précurseur, et un nombre élevé d'AMF. Ce processus très énergivore est finement régulé par des mécanismes qui coordonnent de manière dynamique les niveaux de production de ribosomes aux besoins de la cellule. Ces régulations permettent notamment d'éviter des pertes importantes en énergie cellulaire qui résulteraient d'une production inutile de ribosomes. Au cours de mon doctorat, j'ai étudié des évènements précis de régulation de la biogenèse des ribosomes orchestrés par la voie de signalisation Ras-MAPK/ERK. Cette voie de signalisation est l'un des principaux acteurs de la croissance et de la prolifération cellulaire. ERK et sa kinase effectrice RSK stimulent trois événements clés de la biogénèse des ribosomes : la transcription Pol I/Pol III, le transport nucléo-cytoplasmique et la traduction. Cependant, aucun substrat de cette voie n'a été clairement identifié dans les étapes post-transcriptionnelles de la biogenèse des ribosomes, à savoir les étapes d'assemblage et de maturation des ribosomes. Mes travaux ont permis d'identifier la kinase atypique RIOK2 comme une nouvelle cible de la kinase effectrice RSK. Nous avons découvert que la phosphorylation de RIOK2 par RSK favorise sa dissociation des particules pré-40S, facilitant ainsi la production de la particule mature 40S. Outre ces résultats, nous avons également identifié et caractérisé des partenaires proches de RIOK2 dans la cellule. Cette analyse ouvre la voie vers de nouvelles connexions entre RIOK2, ou d'autres AMF, et des processus intracellulaires clés autres que la synthèse des ribosomes. Dans l'ensemble, ma thèse aura contribué à la découverte de nouvelles perspectives de recherche dans la régulation des étapes de maturation des ribosomes. L'identification de nouveaux mécanismes de régulation pourrait aider à mieux intégrer les phénotypes associés à des dérégulations de la biogenèse des ribosomes, en conditions physiologiques ou pathologiquesRibosome biogenesis feeds the cellular needs in protein synthesis by synthetizing translation-competent ribosomes. This highly energy-consuming process mobilizes the three RNA polymerases (Pol) and the translational machinery, active import and export through the nucleo-cytoplasmic network, as well as an intricate maturation pathway that involves more than 200 assembly and maturation factors (AMFs). In proliferating mammalian cells, the synthesis rate of ribosomes has been estimated at 7500 ribosomal subunits per minutes, requiring ~300 000 ribosomal proteins (RPs), 150 small nucleolar RNAs (snoRNAs) per pre-rRNA and an even higher number of AMFs. This fuel-consuming cellular process is tightly regulated by mechanisms that dynamically coordinate ribosome levels with cell requirements, thereby preventing energy waste due to production of unnecessary ribosomes. During my thesis, I studied discrete ribosome biogenesis regulatory events orchestrated by the Ras-MAPK/ERK signaling pathway. This signaling pathway is one of the main actor of cell growth and proliferation. ERK and its downstream effector kinase RSK stimulate three key events of ribosome biogenesis: Pol I/Pol III transcription, nucleo-cytoplasmic transport, and translation. However, no substrate of this pathway has been clearly identified in the post-transcriptional steps of ribosome biogenesis, namely ribosome assembly and maturation. My study identified the kinase RIOK2 as a new target of RSK kinase. We found that phosphorylation of RIOK2 by RSK promotes its dissociation from pre-40S particles, thereby facilitating the completion of small ribosomal subunit synthesis. Beside these findings, we have characterized the RIOK2 proximal interactome. Analysis of the proteins spatially close to RIOK2 paves the way to new connections between RIOK2, as well as other AMFs, and key intracellular processes other than ribosome biogenesis. Altogether, my thesis contributed to the discovery of novel insights into the regulation of ribosome maturation steps. Identification of novel regulatory events may help better integrating phenotypes associated with deregulation of ribosome biogenesis, during both physiological changes and diseases
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Kihara, Akio. "Identification,characterization,and regulation of the proteolytic system that degrades uncomplexed SecY subunit of protein translocase in the Escherichia coli plasma membrane." 京都大学 (Kyoto University), 1998. http://hdl.handle.net/2433/157158.
Повний текст джерелаАнотація:
本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(理学)
甲第7164号
理博第1938号
新制||理||1043(附属図書館)
UT51-98-G93
京都大学大学院理学研究科化学専攻
(主査)教授 伊藤 維昭, 教授 井上 丹, 教授 三木 邦夫
学位規則第4条第1項該当
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Sardana, Richa. "From knobs to a central pseudoknot : understanding 40S ribosomal subunit biogenesis through Bud23." Thesis, 2013. http://hdl.handle.net/2152/30458.
Повний текст джерелаАнотація:
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
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Kouba, Tomáš. "Mapování kontaktních míst mezi eukaryotickým translačním iniciačním faktorem eIF3 a 40S ribozomální podjednotkou." Doctoral thesis, 2013. http://www.nusl.cz/ntk/nusl-329277.
Повний текст джерелаАнотація:
Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs) together with the small ribosomal subunit to locate the mRNA's translational start and to properly decode the genetic message that it carries. The largest of these factors, eIF3, forms the scaffold for other initiation factors to promote their spatially coordinated placement on the ribosomal surface. It is our long-standing pursuit to map the 40S-binding site of the yeast multisubunit eIF3 and here we present three new mutual interactions between these two macromolecules (i) The C-terminal region of the eIF3c/NIP1 subunit is comprised of the conserved bipartite PCI domain and we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 in vivo. The extreme C-terminus directly interacts with small subunit ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of ASC1 impairs eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this protein fold in protein-RNA interactions. We conclude that the c/NIP1...
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White, Joshua Paul 1977. "The role of Bud23 in the biogenesis of the small ribosomal subunit in Saccharomyces cerevisiae." Thesis, 2009. http://hdl.handle.net/2152/ETD-UT-2009-08-331.
Повний текст джерелаАнотація:
Ribosomes are the cellular structures responsible for the synthesis of protein in all branches of life. All ribosomes are made from a large and small subunit that in turn are composed of protein and RNA. The synthesis of eukaryotic ribosomes is a complex process involving more than 200 factors and spans three cellular compartments: the nucleolus, the nucloplasm, and the cytoplasm. The precise function of most of these ribosome biogenesis factors remains unknown. The RNA component of ribosomes is in part processed from a large RNA transcript that yields most of the RNA present in mature ribosomes. Part of the maturation process involves modification of this ribosomal RNA as processing is carried out.
Recent work constructing protein interaction networks in Saccharomyces cerevisiae suggested the methyltransferase Bud23 was involved in ribosome biogenesis (1). This thesis describes my work to characterize Bud23 and place it within the ribosome biogenesis pathway. Bud23 is a SAM methyltransferase important for the proper biogenesis of the small ribosomal subunit. Here I will demonstrate that Bud23 methylates G1575 of the small subunit ribosomal RNA (SSU rRNA), and its absence delays export of the SSU rRNA from the nucleolas, and the nucleus, and results in the delayed maturation of the SSU rRNA. Finally, I will show that Bud23 function is connected to small subunit processome factor Utp14 through identification of a Utp14 mutant that suppresses the bud23[Delta] deletion phenotype.text
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Vlčková, Vladislava. "Úloha N-terminální domény a/TIF32 podjednotky iniciačního faktoru eIF3 ve vazbě mRNA na 43S pre-iniciační komplexy." Master's thesis, 2013. http://www.nusl.cz/ntk/nusl-321098.
Повний текст джерелаАнотація:
Translation initiation is a complex process which results in the assembly of the elongation competent 80S ribosome from the 40S and 60S ribosomal subunits, the initiator tRNA and mRNA, and is orchestrated by numerous eukaryotic initiation factors (eIFs). Although it represents one of the most regulated processes of gene expression, the exact mechanism of one of the key steps of translation initiation - mRNA recruitment to the 43S pre-initiation complex (PIC) - is still only poorly understood. Recent studies indicated that besides eIF4F and poly(A)-binding protein, also eIF3 might play an important, if not crucial, role in this step. In our laboratory, we recently identified a 10 Ala substitution (Box37) in the a/TIF32 subunit of Saccharomyces cerevisiae eIF3, which interfered with translation initiation rates. Detailed analysis showed that this mutation significantly reduces the amounts of model mRNA in the gradient fractions containing 48S PICs as the only detectable effect in vivo. Moreover, a recently solved crystal structure of the N-terminal part of a/TIF32 pointed to two Box37 residues, Arg363 and Lys364, both proposed to contribute to one of the positive, potentially RNA-binding areas on the a/TIF32 surface. The fact that also their substitutions with alanines severely impaired the mRNA recruitment...
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Jobe, Amy Beth. "Cryo-electron microscopy and single particle reconstructions of the Leishmania major ribosome and of the encephalomyocarditis virus internal ribosome entry site bound to the 40S subunit." Thesis, 2017. https://doi.org/10.7916/D85T3R4W.
Повний текст джерелаАнотація:
The ribosome is a macromolecular machine, present in high copy number in the cell, that synthesizes proteins from information encoded in messenger RNA. It is a universal translator, found in all life forms and in all eras recent enough to bear life. The ribosome is structurally complex and its structure is highly evolutionarily conserved; that conservation reinforces the concept that its function in executing translation is essential. As a subject of study, the ribosome lends itself well to direct imaging, as it is large, asymmetric, dynamic, and it interacts with other heterogeneous agents throughout the translation process; if we are to infer function from structure, then the most certain way to observe the ribosome’s structure is to image it as directly as possible. Cryo-electron microscopy and single particle reconstruction are appropriate tools for this endeavor, as they can produce high-resolution three-dimensional structures of ribosomes or other macromolecular samples, and they can even reveal multiple biologically relevant states of a single sample.
Although the ribosome is highly conserved in terms of its presence and core structure and functions, there is considerable variation among taxa, and the function of some of this variation is not yet understood. For example, the ribosome of the unicellular trypanosomatid parasite Leishmania major exhibits unusually large expansion segments of ribosomal RNA, as well as unusual cleavage sites in ribosomal RNA that is otherwise conserved. Here, we present a three-dimensional cryo-electron microscopy reconstruction of the 80S ribosome of Leishmania major and compare it to the available ribosome structures of closely related parasites.
There is also structural variation related to the mechanism of translation: certain viruses with RNA genomes employ highly structured segments of RNA called internal ribosome entry sites to initiate translation of viral proteins on host cell ribosomes via noncanonical mechanisms. We explore one instance of this with a reconstruction of the encephalomyocarditis virus internal ribosome entry site bound with necessary protein factors to a eukaryotic 40S ribosomal subunit.
Частини книг з теми "40S subunit"
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Spahn, Christian M. T., Jeffrey S. Kieft, Robert A. Grassucci, Pawel A. Penczek, Kaihong Zhou, Jennifer A. Doudna, and Joachim Frank. "Hepatitis C Virus IRES RNA–Induced Changes in the Conformation of the 40S Ribosomal Subunit." In Series in Structural Biology, 300–303. World Scientific, 2018. http://dx.doi.org/10.1142/9789813234864_0028.
Повний текст джерела
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Frank, Joachim. "Computer Averaging of Electron Micrographs of 40S Ribosomal Subunits." In Series in Structural Biology, 109–11. World Scientific, 2018. http://dx.doi.org/10.1142/9789813234864_0009.
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"Returning." In The Daode jing Commentary of Cheng Xuanying, translated by Friederike Assandri, 206–7. Oxford University Press, 2021. http://dx.doi.org/10.1093/oso/9780190876456.003.0041.
Повний текст джерелаАнотація:
This chapter is the translation of chapter 40 of the Expository Commentary to the Daode jing by Cheng Xuanying. Cheng reads the ancient Daode jing in the light of early Tang dynasty Daoism. In the early medieval period up to the Tang, Daoism had developed in close contact with Buddhism, adopting and co-opting many concepts and ideas from Buddhism. Cheng brings these originally Buddhist concepts into the exegesis of the classic Daode jing. In addition, he ties the Daode jing closely to the Zhuangzi and to Confucian classics by citing them in his commentary to every chapter of the Daode jing. He also uses the Buddhist method of kepan to structure the text of the single chapter into smaller subunits and also to create a framework explaining the specific sequence of the single chapters in the Daode jing.
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Zhan, Xianquan, and Miaolong Lu. "Abnormal Ubiquitination of Ubiquitin-Proteasome System in Lung Squamous Cell Carcinomas." In Ubiquitin - Proteasome Pathway. IntechOpen, 2020. http://dx.doi.org/10.5772/intechopen.93586.
Повний текст джерелаАнотація:
Ubiquitination is an important post-translational modification. Abnormal ubiquitination is extensively associated with cancers. Lung squamous cell carcinoma (LUSC) is the most common pathological type of lung cancer, with unclear molecular mechanism and the poor overall prognosis of LUSC patient. To uncover the existence and potential roles of ubiquitination in LUSC, label-free quantitative ubiquitomics was performed in human LUSC vs. control tissues. In total, 627 ubiquitinated proteins (UPs) with 1209 ubiquitination sites were identified, including 1133 (93.7%) sites with quantitative information and 76 (6.3%) sites with qualitative information. KEGG pathway enrichment analysis found that UPs were significantly enriched in ubiquitin-mediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). Further analysis of 400 differentially ubiquitinated proteins (DUPs) revealed that 11 subunits of the proteasome complex were differentially ubiquitinated. These findings clearly demonstrated that ubiquitination was widely present in the ubiquitin-proteasome pathway in LUSCs. At the same time, abnormal ubiquitination might affect the function of the proteasome to promote tumorigenesis and development. This book chapter discussed the status of protein ubiquitination in the ubiquitin-proteasome system (UPS) in human LUSC tissues, which offered the scientific data to elucidate the specific molecular mechanisms of abnormal ubiquitination during canceration and the development of anti-tumor drugs targeting UPS.
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Kagendo, Dorothy, Eric Muchiri, Peter Gitonga, and Esther Muthoni. "Interlinks between Wildlife and Domestic Cycles of Echinococcus spp. in Kenya." In Managing Wildlife in a Changing World [Working Title]. IntechOpen, 2020. http://dx.doi.org/10.5772/intechopen.94612.
Повний текст джерелаАнотація:
Effective conservation and management of wildlife in the current changing world, call for incorporation of infectious zoonotic diseases surveillance systems, among other interventions. One of such diseases is echinococcosis, a zoonotic disease caused by Echinococcus species. This disease exists in two distinct life cycle patterns, the domestic and wildlife cycles. To investigate possible inter-links between these cycles in Kenya, 729 fecal samples from wild carnivores and 406 from domestic dogs (Canis lupus familiaris) collected from Maasai Mara and Samburu National Reserves were analyzed. Taeniid eggs were isolated by zinc chloride sieving-flotation method and subjected to polymerase chain reaction of nicotinamide adenine dehydrogenase subunit 1 (NAD1). Subsequent amplicons were sequenced, edited and analyzed with GENtle VI.94 program. The samples were further subjected to molecular identification of specific host species origin. All sequences obtained were compared with those in Gene-bank using Basic Local Alignment Search Tool (BLAST). The study found that there were 74 taeniid positive samples, 53 from wild carnivores and 21 from domestic dogs. In wildlife, mixed infections with Echinococcus and Taenia species were identified and these included E. granulosus sensu stricto, E. felidis, T. canadensis G6/7, Taenia hydatigena, T. multiceps, and T. saginata. Domestic dogs harbored Echinococcus and Taenia species similar to wild carnivores including E. granulosus G1–3, E. felidis, T. multiceps, T. hydatigena, and T. madoquae. Taenia species of nine taeniid eggs were not identified. Majority of genotypes were found in hyena (Crocuta crocuta) fecal samples. Distribution of Echinococcus and Taenia spp. varied with hosts. Mixed infections of Echinococcus spp, T. multiceps and T. hydatigena in a single animal were common. There seemed to be existence of interactions between the two cycles, although public health consequences are unknown. The presence of T. saginata in hyena suggests scavenging of human fecal matter by the animal. In addition, presence of T. multiceps, T hydatigena, T madoquae and T. saginata in the two cycles suggested possible human exposure to these parasites. The results are important in drawing up of strategies and policies towards prevention and control of Echinococcosis and other Taenia related parasitic infections, especially in endemic areas given their potential risk to public and socio- economic livelihood.
Тези доповідей конференцій з теми "40S subunit"
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"Phosphomimetically mutated and thus constitutively active kinase of ribosomal protein S6 from Arabidopsis thaliana (AtRPS6K2) does phosphorylate TaRPS6 in wheat (Triticum aestivum) 40S ribosomal subunit." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-214.
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Yoneda, Yusuke, Tetsuro Katayama, Yutaka Nagasawa, Hiroshi Miyasaka, and Yasufumi Umena. "Energy Transfer between Subunits of Photosystem II Dimer Observed by Femtosecond Transient Absorption." In International Conference on Ultrafast Phenomena. Washington, D.C.: OSA, 2016. http://dx.doi.org/10.1364/up.2016.utu4a.40.
Повний текст джерела
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Niiya, K., P. Kostel, T. S. Zimmerman, and Z. M. Ruggeri. "CHARACTERIZATION OF A 40 kDa FRAGMENT OF VON WILLEBRAND FACTOR THAT CONTAINS THE GLYCOPROTEIN IIb/IIIa-BINDING DOMAIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642874.
Повний текст джерелаАнотація:
We have isolated a 40 kDa fragment of von Willebrand factor (vWF) that contains the glycoprotein (GP) Ilb/IIIa-binding domain. The Staphylococcus aureus V8 protease-generated fragment II was digested with trypsin (1:50 enzyme:substrate ratio on a weight-to-weight basis). After addition of a 100fold molar excess of (p-amidinophenyl)methanesulfonyl fluoride in order to inhibit any residual trypsin activity, the whole digest was subjected to ion-exchange and size-exclusion high pressure liquid chromatography. Two major fragments were separated. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) demonstrated that one of the two purified polypeptides had an apparent molecular weight of 40 kDa under both reducing and nonreducing conditions, suggesting that it was a single chain polypeptide. The other fragment had an apparent molecular weight of 22 kDa after reduction and 44 kDa unreduced, suggesting that it was a homodimer. Amino terminal sequence analysis of both fragments was performed by classical Edman degradation following electroelution from reduced SDS-polyacrylamide gels. The amino terminus of the 40 kDa fragment corresponded to residue Glu (1366) (as did the fragment II from which it was derived), while the amino terminus of the 22 kDa fragment corresponded to residue Val (1927) of the constituent 2050 residue subunit. The effect of both fragments on vWF binding to the platelet membrane GP IIb/IIIa complex was evaluated by measuring the residual binding of 125I-labeled vWF to thrombin-stimulated platelets in the presence of varying amounts of the unreduced fragments. The 40 kDa polypeptide inhibited 64 percent of vWF binding when tested at a concentration of 20 μK, whereas the 22kDa dimer was without effect. This study establishes that the GP IIb/IIIa-binding domain of vWF resides in a discrete, single-chain 40 kDa fragment derived from the 220 kDa, homodimeric fragment II generated by V8 protease. Moreover, we found evidence for the existence of inter-chain disulfide bonds within 22 kDa from the carboxyl terminus of the constituent subunit.
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Howard, M. A., M. Coghlan, and B. G. Firkin. "EFFECT OF ELASTASE INDUCED CLEAVAGE OF VON WILLEBRAND FACTOR (vWf) ON ITS STRUCTURE AND FUNCTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644091.
Повний текст джерелаАнотація:
A proteolytic product of vWf termed fast migrating protein (vWf:FMP) has been identified using crossed immunoelectrophoresis (CIE) in normal serum and in the plasma from patients with disseminated intravascular coagulation (DIC). A fragment of vWf antigen (vWf:Ag) migrating to a similar position on CIE as vWf:FMP results from digestion of vWf:Ag with polymorphonuclear cells (PMNC). Since parallels exist between the conditions for generation of vWf:FMP by PMNC and elastase release by these cells the effect of purified elastase from porcine pancrease on vWf was investigated.VWf was purified from plasma using polyethylene glycol, ammonium sulphate and zinc acetate precipitation, high speed centrifugation and elution from column of Sepharose 4B-CL. A fraction rich in vWf was radiolabelled with 125-Iodine to spike the purified preparation of vWf in order to increase the sensitivity of the protein detecting systems.A mixture of radiolabelled and non-labelled purified vWf was incubated with elastase at concentrations ranging from 2.5 to 40 U/ml for periods of 0-48 hours. Modifications of the structure were assessed by SDS-agarose multimeric analysis, SDS-polyacrylamide electrophoresis and CIE. Alterations of function were quantitated by antigen levels, ristocetin (RCof) and botrocetin (BCoF) cofactor assays and a binding assay to fixed washed platelets in the presence of ristocetin or botrocetin.These investigations show, 1. all but the highest molecular weight multimers of vWf are present when elastase has cleaved vWf such that no intact 240K subunit is present. 2. an intact 240K subunit is not essential for RCoF and BCoF activity or for ristocetin or botrocetin induced binding to platelets.
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Polgár, J., Y. Hidasi, A. Toth, and L. Muszbek. "MEASUREMENT OF FACTOR XIII ACTIVITY IN HUMAN PLATELET HOMOGENATE BY A NEW UV-KINETIC METHOD." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644647.
Повний текст джерелаАнотація:
Factor XIII (FXIIl) of blood coagulation is a zymogen which is converted into an active transglutaminase during the clotting process. Earlier methods used for its determination are cumbersome, laborious, and not suitable for routine laboratory measurements.Most recently we have designed a new simple UV-kinetic assay for the determination of FXIII in the plasma (Muszbek et al., Clin. Chem., 3JL, 35, 1985). The assay is performed on def:j.b-rinated plasma in which FXIII is activated by thrombin and Ca2+. Acetylateddephosphorylated (AD)β-casein and ethylamine are used as substrates and the ammonia released during thereaction is continuously monitored by a NADPH dependent indicator reaction at 340 nm. As the enzymatically active a subunit of FXIII is also present in platelets and monocytes/macrophages we attempted to adapt the above method for the measurement of cellular FXIII activity. Experiments were carried out on Lubrol extract of washed sonicated platelets. It was found that the small amount of fibrinogen present in platelets does not need to be removed and in the blank hirudin used for preventing activation of plasma FXIII should be replaced by EGTA. The concentration of substrates and activators were optimized. The methodwas found linear at least up-to 40 U/l enzyme activity. It had a good reproducibility (optimal conditionvariance was less than 3%) and correlated well with the most commonly used fluorescent amine (dansylcadaverine) incorporation assay. The method was adapted to a centrifugal fast analyser (Baker, Centrifichem). In addition to congenital FXIII deficiency the determination of FXIII in platelets by this new methodmight have a diagnostic importance in haemopoietic diseases with diminished or accelerated platelet production.
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Dahiback, Bjorn, Ake Lundwall, Andreas Hillarp, Johan Malm, and Johan Stenflo. "STRUCTURE AND FUNCTION OF VITAMIN K-DEPENDENT PROTEIN S, a cofactor to activated protein C which also interacts with the complement protein C4b-binding protein." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642960.
Повний текст джерелаАнотація:
Protein S is a single chain (Mr 75.000) plasma protein. It is a cofactor to activated protein C (APC) in the regulation of coagulation factors Va and Villa. It has high affinity for negatively charged phospolipids and it forms a 1:1 complex with APC on phospholipid surfaces, platelets and on endothelial cells. Patients with heterozygous protein S deficiency have a high incidence of thrombosis. Protein S is cleaved by thrombin, which leads to a loss of calcium binding sites and of APC cofactor activity. Protein S has two to three high affinity (KD 20uM) calcium binding sites - unrelated to the Gla-region - that are unaffected by the thrombin cleavage. In human plasma protein S (25 mg/liter) circulates in two forms; free (approx. 40%) and in a 1:1 noncovalent complex (KD 1× 10-7M) with the complement protein C4b-binding protein (C4BP). C4BP (Mr 570.000) is composed of seven identical 70 kDa subunits that are linked by disulfide bonds. When visualized by electron microscopy, C4BP has a spiderlike structure with the single protein S binding site located close to the central core and one C4b-binding site on each of the seven tentacles. When bound to C4BP, protein S looses its APC cofactor activity, whereas the function-of C4BP is not directly affected by the protein S binding. Chymotrypsin cleaves each of the seven C4BP subunits close to the central core which results in the liberation of multiple 48 kDa “tentacte” fragments and the formation of a 160 kDa central core fragment. We have successfully isolated a 160 kDa central core fragment with essentially intact protein S binding ability.The primary structure of both bovine and human protein S has been determined and found to contain 635 and 634 amino acids, respectively, with 82 % homology to each other. Four different regions were distinguished; the N-terminal Gla-domain (position 1-45) was followed by a region which has two thrombin-sensitive bonds positioned within a disulfide loop. Position 76 to 244 was occupied by four repeats homologous to the epidermal growth factor (EGF) precursor. In the first EGF-domain a modified aspartic acid was identified at position 95, B-hydroxaspartic acid (Hya), and in corresponding positions in the three following EGF-domains (positions 136,178 and 217) we found B-hydroxyasparagine (Hyn). Hyn has not previously been identified in proteins. The C-terminal half of protein S (from position 245) shows no homology to the serine proteases but instead to human Sexual Hormon Binding Globulin (SHBG)(see separate abstract). To study the structure-function relationship we made eighteen monoclonal antibodies to human protein S. The effects of the monoclonals on the C4BP-protein S interaction and on the APC cofactor activity were analysed. Eight of the antibodies were calciumdependent, four of these were against the Gla-domain, two against the thrombin sensitive portion and two against the region bearing the high affinity calcium binding sites. Three of the monoclonals were dependent on the presence of chelating agents, EDTA or EGTA, and were probably directed against the high affinity calcium binding region. Three other monoclonals inhibited the protein S-C4BP interaction. At present, efforts are made to localize the epitopes to gain information about functionally important regions of protein S.
Звіти організацій з теми "40S subunit"
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Chamovitz, Daniel A., and Albrecht G. Von Arnim. eIF3 Complexes and the eIF3e Subunit in Arabidopsis Development and Translation Initiation. United States Department of Agriculture, September 2009. http://dx.doi.org/10.32747/2009.7696545.bard.
Повний текст джерелаАнотація:
The original working hypothesis of our proposal was that The “e” subunit of eIF3 has multiple functions from both within the nucleus and in the cytoplasm. Within this model, we further hypothesized that the “e” subunit of eIF3 functions in translation as a repressor. We proposed to test these hypotheses along the following specific aims: 1) Determine the subcellular localization of the interaction between eIF3e and other eIF3 subunits, or the COP9 signalosome. 2) Elucidate the biological significance of the varied subcellular localizations of eIF3e through generating Arabidopsis eIF3e alleles with altered subcellular localization. 3.) Purify different eIF3e complexes by tandem affinity purification (TAP). 4) Study the role of eIF3e in translational repression using both in vitro and in planta assays. eIF3 is an evolutionarily ancient and essential component of the translational apparatus in both the plant and animal kingdoms. eIF3 is the largest, and in some ways the most mysterious, of the translation factors. It is a multi-subunit protein complex that has a structural/scaffolding role in translation initiation. However, despite years of study, only recently have differential roles for eIF3 in the developmental regulation of translation been experimentally grounded. Furthermore, the roles of individual eIF3 subunits are not clear, and indeed some, such as the “e” subunit may have roles independent of translation initiation. The original three goals of the proposal were technically hampered by a finding that became evident during the course of the research – Any attempt to make transgenic plants that expressed eIF3e wt or eIF3e variants resulted in seedling lethality or seed inviability. That is, it was impossible to regenerate any transgenic plants that expressed eIF3e. We did manage to generate plants that expressed an inducible form of eIF3e. This also eventually led to lethality, but was very useful in elucidating the 4th goal of the research (Yahalom et al., 2008), where we showed, for the first time in any organism, that eIF3e has a repressory role in translation. In attempt to solve the expression problems, we also tried expression from the native promoter, and as such analyzed this promoter in transgenic plants (Epel, 2008). As such, several additional avenues were pursued. 1) We investigated protein-protein interactions of eIF3e (Paz-Aviram et al., 2008). 2) The results from goal #4 led to a novel hypothesis that the interaction of eIF3e and the CSN meets at the control of protein degradation of nascent proteins. In other words, that the block in translation seen in csn and eIF3e-overexpressing plants (Yahalom et al., 2008) leads to proteasome stress. Indeed we showed that both over expression of eIF3e and the csn mutants lead to the unfolded protein response. 3) We further investigated the role of an additional eIF3 subunit, eIF3h, in transalational regulation in the apical meristem (Zhou et al., 2009). Epel, A. (2008). Characterization of eIF3e in the model plant Arabidopsis thaliana. In Plant Sciences (Tel Aviv, Tel Aviv University). Paz-Aviram, T., Yahalom, A., and Chamovitz, D.A. (2008). Arabidopsis eIF3e interacts with subunits of the ribosome, Cop9 signalosome and proteasome. Plant Signaling and Behaviour 3, 409-411. Yahalom, A., Kim, T.H., Roy, B., Singer, R., von Arnim, A.G., and Chamovitz, D.A. (2008). Arabidopsis eIF3e is regulated by the COP9 signalosome and has an impact on development and protein translation. Plant J 53, 300-311. Zhou, F., Dunlap, J.R., and von Arnim, A.G. The translation initiation factor subunit eIF3h is .1 involved in Arabidopsis shoot apical meristem maintenance and auxin response. (submitted to Development).
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Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.
Повний текст джерелаАнотація:
The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.
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Dickman, Martin B., and Oded Yarden. Phosphorylative Transduction of Developmental and Pathogenicity-Related Cues in Sclerotinia Sclerotiorum. United States Department of Agriculture, April 2004. http://dx.doi.org/10.32747/2004.7586472.bard.
Повний текст джерелаАнотація:
Sclerotinia sclerotiorum (Lib.) de Bary is among the world's most successful and omnivorous fungal plant pathogens. Included in the more than 400 species of plants reported as hosts to this fungus are canola, alfalfa, soybean, sunflower, dry bean, and potato. The general inability to develop resistant germplasm with these economically important crops to this pathogen has focused attention on the need for a more detailed examination of the pathogenic determinants involved in disease development. This proposal involved experiments that examined the involvement of protein phosphorylation during morphogenesis (hyphal elongation and sclerotia formation) and pathogenesis (oxalic acid). Data obtained from our laboratories during the course of this project substantiates the fact that kinases and phosphatases are involved and important for these processes. A mechanistic understanding of the successful strategy(ies) used by S . sclerotiorum in infecting and proliferating in host plants and this linkage to fungal development will provide targets and/or novel approaches with which to design resistant crop plants including interference with fungal pathogenic development. The original objectives of this grant included: I. Clone the cyclic AMP-dependent protein kinase A (PKA) catalytic subunit gene from S.sclerotiorum and determine its role in fungal pathogenicity, OA production (OA) and/or morphogenesis (sclerotia formation). II. Clone and characterize the catalytic and regulatory subunits of the protein phosphatase PP2A holoenzyme complex and determine their role in fungal pathogenicity and/or morphogenesis as well as linkage with PKA-regulation of OA production and sclerotia formation. III. Clone and characterize the adenylate cyclase-encoding gene from S . sclerotiorum and detennine its relationship to the PKA/PP2A-regulated pathway. IV. Analyze the expression patterns of the above-mentioned genes and their products during pathogenesis and determine their linkage with infection and fungal growth.
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Gelb, Jr., Jack, Yoram Weisman, Brian Ladman, and Rosie Meir. Identification of Avian Infectious Brochitis Virus Variant Serotypes and Subtypes by PCR Product Cycle Sequencing for the Rational Selection of Effective Vaccines. United States Department of Agriculture, December 2003. http://dx.doi.org/10.32747/2003.7586470.bard.
Повний текст джерелаАнотація:
Objectives 1. Determine the serotypic identities of 40 recent IBV isolates from commercial chickens raised in the USA and Israel. 2. Sequence all IBV field isolates using PCR product cycle sequencing and analyze their S 1 sequence to detennine their homology to other strains in the Genbank and EMBL databases. 3. Select vaccinal strains with the highest S 1 sequence homology to the field isolates and perform challenge of immunity studies in chickens in laboratory trials to detennine level of protection afforded by the vaccines. Background Infectious bronchitis (IB) is a common, economically important disease of the chicken. IB occurs as a respiratory form, associated with airsacculitis, condemnation, and mortality of meat-type broilers, a reproductive form responsible for egg production losses in layers and breeders, and a renal form causing high mortality in broilers and pullets. The causative agent is avian coronavirus infectious bronchitis virus (IBV). Replication of the virus' RNA genome is error-prone and mutations commonly result. A major target for mutation is the gene encoding the spike (S) envelope protein used by the virus to attach and infect the host cell. Mutations in the S gene result in antigenic changes that can lead to the emergence of variant serotypes. The S gene is able to tolerate numerous mutations without compromising the virus' ability to replicate and cause disease. An end result of the virus' "flexibility" is that many strains of IBV are capable of existing in nature. Once formed, new mutant strains, often referred to as variants, are soon subjected to immunological selection so that only the most antigenically novel variants survive in poultry populations. Many novel antigenic variant serotypes and genotypes have been isolated from commercial poultry flocks. Identification of the field isolates of IBV responsible for outbreaks is critical for selecting the appropriate strain(s) for vaccination. Reverse transcriptase polymerase chain reaction (RT-PCR) of the Sl subunit of the envelope spike glycoprotein gene has been a common method used to identify field strains, replacing other time-consuming or less precise tests. Two PCR approaches have been used for identification, restriction fragment length polymorphism (RFLP) and direct automated cycle sequence analysis of a diagnostically relevant hypervariab1e region were compared in our BARD research. Vaccination for IB, although practiced routinely in commercial flocks, is often not protective. Field isolates responsible for outbreaks may be unrelated to the strain(s) used in the vaccination program. However, vaccines may provide varying degrees of cross- protection vs. unrelated field strains so vaccination studies should be performed. Conclusions RFLP and S1 sequence analysis methods were successfully performed using the field isolates from the USA and Israel. Importantly, the S1 sequence analysis method enabled a direct comparison of the genotypes of the field strains by aligning them to sequences in public databases e.g. GenBank. Novel S1 gene sequences were identified in both USA and Israel IBVs but greater diversity was observed in the field isolates from the USA. One novel genotype, characterized in this project, Israel/720/99, is currently being considered for development as an inactivated vaccine. Vaccination with IBV strains in the US (Massachusetts, Arkansas, Delaware 072) or in Israel (Massachusetts, Holland strain) provided higher degrees of cross-protection vs. homologous than heterologous strain challenge. In many cases however, vaccination with two strains (only studies with US strains) produced reasonable cross-protection against heterologous field isolate challenge. Implications S1 sequence analysis provides numerical similarity values and phylogenetic information that can be useful, although by no means conclusive, in developing vaccine control strategies. Identification of many novel S1 genotypes of IBV in the USA is evidence that commercial flocks will be challenged today and in the future with strains unrelated to vaccines. In Israel, monitoring flocks for novel IBV field isolates should continue given the identification of Israel/720/99, and perhaps others in the future. Strains selected for vaccination of commercial flocks should induce cross- protection against unrelated genotypes. Using diverse genotypes for vaccination may result in immunity against unrelated field strains.