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Sample records for 30s ribosomal subunits

  1. An assembly landscape for the 30S ribosomal subunit

    PubMed Central

    Talkington, Megan W. T.; Siuzdak, Gary

    2005-01-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions. PMID:16319883

  2. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

    SciTech Connect

    Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; Lemke, Henrik T.; Alonso-Mori, Roberto; Coey, Aaron; Larsen, Kevin; Baxter, Elizabeth L.; Cohen, Aina E.; Soltis, S. Michael; DeMirci, Hasan

    2015-04-30

    In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  3. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

    DOE PAGES

    Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; ...

    2015-04-30

    In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecondmore » X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.« less

  4. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  5. Neutron scattering and the 30 S ribosomal subunit of E. coli

    SciTech Connect

    Moore, P.B.; Engelman, D.M.; Langer, J.A.; Ramakrishnan, V.R.; Schindler, D.G.; Schoenborn, B.P.; Sillers, I.Y.; Yabuki, S.

    1982-01-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today. 30 references, 5 figures.

  6. Characteristic views of E. coli and B. stearothermophilus 30S ribosomal subunits in the electron microscope.

    PubMed Central

    van Heel, M; Stöffler-Meilicke, M

    1985-01-01

    Large sets of electron microscopic images of the 30S ribosomal subunits of Bacillus stearothermophilus (914 molecules) and Escherichia coli (422 molecules) were analysed with image processing techniques. Using computer alignment and a new multivariate statistical classification scheme, three predominant views of the subunit were found for both species. These views, which together account for approximately 90% of the population of images, were determined to a reproducible resolution of up to 1.7 nm, thus elucidating many new structural details. The angular spread of the molecular orientations around the three main stable positions is remarkably small (less than 8 degrees). Some of the current models for the small ribosomal subunit are incompatible with our new results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3908096

  7. Crystal Structure of the 30S Ribosomal Subunit from Thermus Thermophilus. Purification, Crystallization and Structure Determination

    SciTech Connect

    Clemons, William M.; Brodersen, Ditlev E.; McCutcheonn, John P.; May, Joanna L.C.; Carter, Andrew P.; Morgan-Warren, Robert J.; Wimberly, Brian T.; Ramakrishnan, Venki

    2009-10-07

    We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 {angstrom} resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 {angstrom} resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

  8. Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

    PubMed Central

    Nord, Stefan; Bhatt, Monika J.; Tükenmez, Hasan; Farabaugh, Philip J.; Wikström, P. Mikael

    2015-01-01

    The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA. PMID:26089326

  9. Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser

    PubMed Central

    Demirci, Hasan; Sierra, Raymond G.; Laksmono, Hartawan; Shoeman, Robert L.; Botha, Sabine; Barends, Thomas R. M.; Nass, Karol; Schlichting, Ilme; Doak, R. Bruce; Gati, Cornelius; Williams, Garth J.; Boutet, Sébastien; Messerschmidt, Marc; Jogl, Gerwald; Dahlberg, Albert E.; Gregory, Steven T.; Bogan, Michael J.

    2013-01-01

    High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes. PMID:23989164

  10. Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser.

    PubMed

    Demirci, Hasan; Sierra, Raymond G; Laksmono, Hartawan; Shoeman, Robert L; Botha, Sabine; Barends, Thomas R M; Nass, Karol; Schlichting, Ilme; Doak, R Bruce; Gati, Cornelius; Williams, Garth J; Boutet, Sébastien; Messerschmidt, Marc; Jogl, Gerwald; Dahlberg, Albert E; Gregory, Steven T; Bogan, Michael J

    2013-09-01

    High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.

  11. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

    PubMed Central

    Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2014-01-01

    Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

  12. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  13. The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway.

    PubMed

    Zarubica, Tamara; Baker, Matthew R; Wright, H Tonie; Rife, Jason P

    2011-02-01

    Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+² dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+² (translationally inactive) and high Mg+² (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.

  14. Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly.

    PubMed

    Holmes, Kristi L; Culver, Gloria M

    2005-11-25

    Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.

  15. Concurrent Nucleation of 16S Folding and Induced Fit in 30S Ribosome Assembly

    SciTech Connect

    Adilakshmi, T.; Bellur, D; Woodson, S

    2008-01-01

    Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting3 was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.

  16. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  17. Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments.

    PubMed

    Yusupov, M M; Spirin, A S

    1986-03-03

    The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.

  18. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  19. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  20. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  1. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  2. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  3. Insights into structure and function of 30S Ribosomal Protein S2 (30S2) in Chlamydophila pneumoniae: A potent target of pneumonia.

    PubMed

    Koteswara Reddy, G; Nagamalleswara Rao, K; Yarrakula, Kiran

    2017-02-01

    The gene 30S ribosomal protein S2 (30S2) is identified as a potential drug and vaccine target for Pneumonia. Its structural characterization is an important to understand the mechanism of action for identifying its receptor and/or other binding partners. The comparative genomics and proteomics studies are useful for structural characterization of 30S2 in C. Pneumoniae using different bioinformatics tools and web servers. In this study, the protein 30S2 structure was modelled and validated by Ramachandran plot. It is found that the modelled protein under most favoured "core" region was 88.7% and overall G-factor statistics with average score was -0.20. However, seven sequential motifs have been identified for 30S2 with reference codes (PR0095, PF0038, TIGR01012, PTHR11489, SSF52313 and PTHR11489). In addition, seven structural highly conserved residues have been identified in the large cleft are Lys160, Gly161and Arg162 with volume 1288.83Å(3) and average depth of the cleft was 10.75Å. Moreover, biological functions, biochemical process and structural constituents of ribosome are also explored. The study will be helped us to understand the sequential, structural, functional and evolutionary clues of unknown proteins available in C. Pneumoniae.

  4. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  5. Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.

    PubMed

    MacDougall, Daniel D; Gonzalez, Ruben L

    2015-05-08

    Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

  6. Effects of cations and cosolvents on eukaryotic ribosomal subunit conformation

    SciTech Connect

    Moore, M.N.; Spremulli, L.L.

    1985-01-01

    The effects of various cations and cosolvents on the conformation of wheat germ ribosomes and ribosomal subunits have been investigated by using the techniques of circular dichroism and differential hydrogen exchange. A class of hydrogens on both the 40S and 60S subunits exchange out more rapidly as the Mg/sup 2 +/ concentration is raised, indicating that Mg/sup 2 +/ causes a change in subunit conformation. Ca/sup 2 +/ and the polyamines produce a similar increase in the rate of hydrogen exchange. These results suggest that increases in cation concentrations permit a tightening of ribosome structure and a greater degree of internalization of the rRNA. The cosolvent glycerol causes an alteration in the CD spectrum of 80S ribosomes in both the nucleic acid and protein portions of the spectrum. Glycerol also causes a decrease in the rate of exchange of a number of hydrogens on both the 40S and 60S subunits. These results are interpreted to mean that glycerol favors a more native, less denatured structure in the ribosome.

  7. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  8. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  9. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  10. Ribosome Subunit Stapling for Orthogonal Translation in E.  coli.

    PubMed

    Fried, Stephen D; Schmied, Wolfgang H; Uttamapinant, Chayasith; Chin, Jason W

    2015-10-19

    The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

  11. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  12. Structure of the large ribosomal subunit from human mitochondria

    PubMed Central

    Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C.; Murshudov, Garib; Scheres, Sjors H. W.; Ramakrishnan, V.

    2014-01-01

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases, and are often the unintended targets of various clinically useful antibiotics. Using single-particle electron cryo-microscopy we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance including recruitment of mitochondrial tRNAVal to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs. PMID:25278503

  13. Structure of the large ribosomal subunit from human mitochondria.

    PubMed

    Brown, Alan; Amunts, Alexey; Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C; Murshudov, Garib; Scheres, Sjors H W; Ramakrishnan, V

    2014-11-07

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

  14. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

    PubMed Central

    Gao, Ning; Zavialov, Andrey V.; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    Summary After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-hydrolysis dependent manner. Based on a previous cryo-electron microscopy (cryo-EM) study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF containing post-termination ribosome triggers an inter-domain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9 Å (FSC at 0.5 cutoff) cryo-EM map of a 50S EFG GDPNP RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the non-cleavable GTP analogue GDPNP. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in tRNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G’s domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits. PMID:17996252

  15. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  16. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  17. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  18. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  19. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  20. Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast.

    PubMed

    Konikkat, Salini; Woolford, John L

    2017-01-15

    Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

  1. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

    PubMed Central

    Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

    2016-01-01

    Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

  2. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    RNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.

  3. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  4. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  5. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli.

    PubMed

    Wagenknecht, T; Grassucci, R; Frank, J

    1988-01-05

    Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.

  6. Purification of 70S ribosomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

  7. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.

    PubMed

    Strunk, Bethany S; Novak, Megan N; Young, Crystal L; Karbstein, Katrin

    2012-07-06

    Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

  8. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  9. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  10. High resolution structure of the large ribosomal subunit from a Mesophilic Eubacterium

    SciTech Connect

    Harms, Joerg; Schluenzen, Frank; Zarivach, Raz; Bashan, Anat; Gat, Sharon; Agmon, Ilana; Bartels, Heike; Franceschi, Francois; Yonath, Ada

    2009-10-07

    We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

  11. Structures of Triacetyloleandomycin and Mycalamide A Bound to the Large Ribosomal Subunit of Haloarcula marismortui

    SciTech Connect

    Gurel, G.; Blaha, G; Steitz, T; Moore, P

    2009-01-01

    Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the large ribosomal subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.

  12. Structures of Triacetyloleandomycin and Mycalamide A Bind to the Large Ribosomal Subunit of Haloarcula marismortui

    SciTech Connect

    Gürel, Güliz; Blaha, Gregor; Steitz, Thomas A.; Moore, Peter B.

    2010-01-14

    Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the large ribosomal subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.

  13. Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

    PubMed Central

    Moy, Terence I.; Silver, Pamela A.

    1999-01-01

    After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

  14. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    PubMed

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology.

  15. YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

    PubMed Central

    Ni, Xiaodan; Davis, Joseph H.; Jain, Nikhil; Razi, Aida; Benlekbir, Samir; McArthur, Andrew G.; Rubinstein, John L.; Britton, Robert A.; Williamson, James R.; Ortega, Joaquin

    2016-01-01

    YphC and YsxC are 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 used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit. PMID:27484475

  16. Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

    SciTech Connect

    Ippolito,J.; Kanyo, Z.; Wang, D.; Franceschi, F.; Moore, P.; Steitz, T.; Duffy, E.

    2008-01-01

    The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

  17. Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

    SciTech Connect

    Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul; Pestka, James J.

    2009-06-01

    Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

  18. Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM.

    PubMed

    Shaikh, Tanvir R; Yassin, Aymen S; Lu, Zonghuan; Barnard, David; Meng, Xing; Lu, Toh-Ming; Wagenknecht, Terence; Agrawal, Rajendra K

    2014-07-08

    Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.

  19. Group I introns in small subunit ribosomal DNA of several Phaeosphaeria species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a study of small subunit ribosomal RNA (SSU-rRNA) gene sequences in Phaeosphaeria species, group I introns were found in 9 of 10 P. avenaria f.sp. avenaria (Paa) isolates, 1 of 2 Phaeosphaeria sp. (P-rye) isolates from Polish rye (Sn48-1), 1 Phaeosphaeria sp. from dallis grass (P-dg) (S-93-48) an...

  20. Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

  1. Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

    PubMed Central

    Ohmayer, Uli; Gamalinda, Michael; Sauert, Martina; Ossowski, Julius; Pöll, Gisela; Linnemann, Jan; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Kumcuoglu, Beril; Leger-Silvestre, Isabelle; Faubladier, Marlène; Griesenbeck, Joachim; Woolford, John; Tschochner, Herbert; Milkereit, Philipp

    2013-01-01

    During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed. PMID:23874617

  2. Mutations in RNAs of both ribosomal subunits cause defects in translation termination.

    PubMed Central

    Arkov, A L; Freistroffer, D V; Ehrenberg, M; Murgola, E J

    1998-01-01

    Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants. Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl-tRNA hydrolysis during termination of protein biosynthesis. The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution. Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination. PMID:9482747

  3. Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid.

    PubMed

    Sanamrad, Arash; Persson, Fredrik; Lundius, Ebba G; Fange, David; Gynnå, Arvid H; Elf, Johan

    2014-08-05

    Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

  4. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  5. Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process

    PubMed Central

    1990-01-01

    To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures. PMID:2211825

  6. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  7. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    PubMed Central

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  8. A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

    PubMed

    Wild, Thomas; Horvath, Peter; Wyler, Emanuel; Widmann, Barbara; Badertscher, Lukas; Zemp, Ivo; Kozak, Karol; Csucs, Gabor; Lund, Elsebet; Kutay, Ulrike

    2010-10-26

    The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

  9. Induction of ribosomal subunits misassembly by antisense RNAs to control cell growth.

    PubMed

    Mangiarotti, G

    2000-08-25

    The assembly of ribosomal subunits starting from free ribosomal RNA and protein of Dictyostelium discoideum was induced in vitro in the presence of several oligoribonucleotides complementary to defined sequences of ribosomal RNA. The reconstituted particles had a full complement of ribosomal proteins, but did not function in an in vitro protein synthesis system and were disassembled following interaction with mRNA. The same result was obtained in vivo by fusing the oligodeossiribonucleotides coding for the selected oligoribonucleotides to the promoter of the gene coding for contact site A protein. This gene is expressed only in the first part of development. Transfected growing cells, transferred in developing buffer in the presence of pulses of cAMP, accumulated significant amounts of the oligoribonucleotides. When retransferred to the growth medium, they grew progressively more slowly, until their doubling time doubled, apparently due to the availability of a limiting amount of functional ribosomes. To avoid disassembly of misassembled subunits (G. Mangiarotti et al., 1997, J. Biol. Chem. 272, 27818-27822), two oligoribonucleotides complementary to sequences present at the 5' ends of pre-17S and pre-26S RNAs were also induced to accumulate during early development with the same technique. When transfected cells were retransferred to the growth medium, their rate of growth declined rapidly to zero and cells died, apparently because they were unable to disassemble misassembled ribosomal subunits and avoid their entry into polyribosomes. This technique to perturb protein synthesis, arrest cell growth, and cause cell suicide will be tested in abnormally growing animal cells.

  10. The ancient heart of the ribosomal large subunit: a response to Caetano-Anolles.

    PubMed

    Petrov, Anton S; Williams, Loren Dean

    2015-04-01

    Our recent Accretion Model of ribosomal evolution uses insertion fingerprints and a "trunk-branch" formalism to recapitulate the building up of common core rRNA of the Large Ribosomal Subunit. The Accretion Model is a conservative and natural extension of a method developed by Bokov and Steinberg (Nature 457:977-80, 2009), which confirms the correctness of lower resolution models by Fox and others. In each of these models, the LSU originates with the peptidyl transferase center (PTC), consistent with expectations that the ribosome is the source of defined-sequence functional proteins. In an adjacent note, Caetano-Anolles (J Mol Evol 80:162-165, 2015) disparages the Accretion Model, because it controverts the 'Growth Inferred by Genothermal Ordering' (GIGO) model. GIGO analyzes secondary structures, assigns the origin of the ribosome to a region outside of the PTC, and assumes or deduces that (i) large protein enzymes of defined amino acid sequence predate ribosomal synthesis of proteins, (ii) proteins directly replicate by non-ribosomal mechanisms, (iii) rRNA unfailingly increases in thermodynamic stability over time, and (iv) the Woese and Fox canonical tree of life is mis-rooted. Much of the specific GIGO critique of the Accretion Model is based on confusion about the three-dimensional nature of RNA and trunk-branch polymorphism; the Accretion Model incorporates several types of trunk-branch relationships.

  11. Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

    PubMed Central

    Antoun, Ayman; Pavlov, Michael Y.; Tenson, Tanel

    2004-01-01

    Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is high-lighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix. PMID:15103398

  12. Molecular architecture of the 90S small subunit pre-ribosome

    PubMed Central

    Sun, Qi; Zhu, Xing; Qi, Jia; An, Weidong; Lan, Pengfei; Tan, Dan; Chen, Rongchang; Wang, Bing; Zheng, Sanduo; Zhang, Cheng; Chen, Xining; Zhang, Wei; Chen, Jing; Dong, Meng-Qiu; Ye, Keqiong

    2017-01-01

    Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors. DOI: http://dx.doi.org/10.7554/eLife.22086.001 PMID:28244370

  13. Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota).

    PubMed

    Barroso, Gérard; Sirand-Pugnet, Pascal; Mouhamadou, Bello; Labarère, Jacques

    2003-10-01

    The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

  14. Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

    PubMed Central

    Van de Peer, Y; Rensing, S A; Maier, U G; De Wachter, R

    1996-01-01

    Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA. PMID:8755544

  15. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

    PubMed Central

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N.; May, Kerrie L.; Kahn, Peter C.

    2015-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. PMID:26483409

  16. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.

    PubMed

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N; May, Kerrie L; Kahn, Peter C; Tumer, Nilgun E

    2015-10-19

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.

  17. Inferring the Ancient History of the Translation Machinery and Genetic Code via Recapitulation of Ribosomal Subunit Assembly Orders

    PubMed Central

    Fournier, Gregory P.; Neumann, Justin E.; Gogarten, J. Peter

    2010-01-01

    Universally conserved positions in ribosomal proteins have significant biases in amino acid usage, likely indicating the expansion of the genetic code at the time leading up to the most recent common ancestor(s) (MRCA). Here, we apply this principle to the evolutionary history of the ribosome before the MRCA. It has been proposed that the experimentally determined order of assembly for ribosomal subunits recapitulates their evolutionary chronology. Given this model, we produce a probabilistic evolutionary ordering of the universally conserved small subunit (SSU) and large subunit (LSU) ribosomal proteins. Optimizing the relative ordering of SSU and LSU evolutionary chronologies with respect to minimizing differences in amino acid usage bias, we find strong compositional evidence for a more ancient origin for early LSU proteins. Furthermore, we find that this ordering produces several trends in specific amino acid usages compatible with models of genetic code evolution. PMID:20208990

  18. Inferring the ancient history of the translation machinery and genetic code via recapitulation of ribosomal subunit assembly orders.

    PubMed

    Fournier, Gregory P; Neumann, Justin E; Gogarten, J Peter

    2010-03-01

    Universally conserved positions in ribosomal proteins have significant biases in amino acid usage, likely indicating the expansion of the genetic code at the time leading up to the most recent common ancestor(s) (MRCA). Here, we apply this principle to the evolutionary history of the ribosome before the MRCA. It has been proposed that the experimentally determined order of assembly for ribosomal subunits recapitulates their evolutionary chronology. Given this model, we produce a probabilistic evolutionary ordering of the universally conserved small subunit (SSU) and large subunit (LSU) ribosomal proteins. Optimizing the relative ordering of SSU and LSU evolutionary chronologies with respect to minimizing differences in amino acid usage bias, we find strong compositional evidence for a more ancient origin for early LSU proteins. Furthermore, we find that this ordering produces several trends in specific amino acid usages compatible with models of genetic code evolution.

  19. The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

    PubMed

    Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

    2015-10-01

    One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.

  20. Phylogenetic classification of the frog pathogen Amphibiothecum (Dermosporidium) penneri based on small ribosomal subunit sequencing

    USGS Publications Warehouse

    Feldman, S.H.; Wimsatt, J.H.; Green, D.E.

    2005-01-01

    We determined 1,600 base pairs of DNA sequence in the 18S small ribosomal subunit from two geographically distinct isolates of Dermosporidium penneri. Maximum likelihood and parsimony analysis of these sequences place D. penneri in the order Dermocystida of the class Mesomycetozoea. The 18S rRNA sequences from these two isolates only differ within a single region of 16 contiguous nucleotides. Based on the distant phylogenetic relationship of these organisms to Amphibiocystidium ranae and similarity to Sphaerothecum destruens we propose the organism be renamed Amphibiothecum penneri.

  1. Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits

    PubMed Central

    Pavlov, Michael Y; Zorzet, Anna; Andersson, Dan I; Ehrenberg, Måns

    2011-01-01

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNAi). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNAi, implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNAi and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10−4 for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases. PMID:21151095

  2. Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryo-EM

    PubMed Central

    Chen, Bo; Kaledhonkar, Sandip; Sun, Ming; Shen, Bingxin; Lu, Zonghuan; Barnard, David; Lu, Toh-Ming; Gonzalez, Ruben L.; Frank, Joachim

    2015-01-01

    Ribosomal subunit association is a key checkpoint in translation initiation, but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding and ribosome recycling, are amenable to study with this method. PMID:26004440

  3. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits.

    PubMed

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-09-06

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1-Utp21, Utp12-Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

  4. RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

    SciTech Connect

    Nissen, Poul; Ippolito, Joseph A.; Ban, Nenad; Moore, Peter B.; Steitz, Thomas A.

    2009-10-07

    Analysis of the 2.4-{angstrom} resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2' OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3' terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

  5. The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit.

    PubMed

    Berry, Katherine E; Waghray, Shruti; Doudna, Jennifer A

    2010-08-01

    The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5' untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2'-OMe oligonucleotides.

  6. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

    PubMed Central

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in

  7. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  8. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  9. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

    PubMed Central

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-01-01

    The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. PMID:27422873

  10. Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit.

    PubMed

    Sarkar, Anshuk; Pech, Markus; Thoms, Matthias; Beckmann, Roland; Hurt, Ed

    2016-12-01

    Nuclear export of preribosomal subunits is a key step during eukaryotic ribosome formation. To efficiently pass through the FG-repeat meshwork of the nuclear pore complex, the large pre-60S subunit requires several export factors. Here we describe the mechanism of recruitment of the Saccharomyces cerevisiae RNA-export receptor Mex67-Mtr2 to the pre-60S subunit at the proper time. Mex67-Mtr2 binds at the premature ribosomal-stalk region, which later during translation serves as a binding platform for translational GTPases on the mature ribosome. The assembly factor Mrt4, a structural homolog of cytoplasmic-stalk protein P0, masks this site, thus preventing untimely recruitment of Mex67-Mtr2 to nuclear pre-60S particles. Subsequently, Yvh1 triggers Mrt4 release in the nucleus, thereby creating a narrow time window for Mex67-Mtr2 association at this site and facilitating nuclear export of the large subunit. Thus, a spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit.

  11. A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

    DOE PAGES

    Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

    2015-12-09

    The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

  12. A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

    SciTech Connect

    Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

    2015-12-09

    The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable region of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.

  13. Are stop codons recognized by base triplets in the large ribosomal RNA subunit?

    PubMed

    Liang, Han; Landweber, Laura F; Fresco, Jacques R

    2005-10-01

    The precise mechanism of stop codon recognition in translation termination is still unclear. A previously published study by Ivanov and colleagues proposed a new model for stop codon recognition in which 3-nucleotide Ter-anticodons within the loops of hairpin helices 69 (domain IV) and 89 (domain V) in large ribosomal subunit (LSU) rRNA recognize stop codons to terminate protein translation in eubacteria and certain organelles. We evaluated this model by extensive bioinformatic analysis of stop codons and their putative corresponding Ter-anticodons across a much wider range of species, and found many cases for which it cannot explain the stop codon usage without requiring the involvement of one or more of the eight possible noncomplementary base pairs. Involvement of such base pairs may not be structurally or thermodynamically damaging to the model. However, if, according to the model, Ter-anticodon interaction with stop codons occurs within the ribosomal A-site, the structural stringency which that site imposes on sense codon.tRNA anticodon interaction should also extend to stop codon.Ter-anticodon interactions. Moreover, with Ter-tRNA in place of an aminoacyl-tRNA, for each of the various Ter-anticodons there is a sense codon that can interact with it preferentially by complementary and wobble base-pairing. Both these considerations considerably weaken the arguments put forth previously.

  14. The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome

    PubMed Central

    McCann, Kathleen L; Teramoto, Takamasa; Zhang, Jun; Tanaka Hall, Traci M; Baserga, Susan J

    2016-01-01

    ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease. DOI: http://dx.doi.org/10.7554/eLife.16381.001 PMID:27077951

  15. Analysis of the interaction between bovine mitochondrial 28 S ribosomal subunits and mRNA.

    PubMed

    Farwell, M A; Schirawski, J; Hager, P W; Spremulli, L L

    1996-11-11

    The small subunit of the bovine mitochondrial ribosome forms a tight complex with mRNAs. This [28 S:mRNA] complex forms as readily on circular mRNAs as on linear mRNAs indicating that a free 5' end on the mRNA is not required for the interaction observed. The effects of monovalent cations on the equilibrium association constant and on the forward and reverse rate constants governing this interaction have been determined. Monovalent cations have a strong effect on the forward rate constant. Increasing the KCl concentration from 1 mM to 100 mM reduces kon by nearly 100-fold. Monovalent cations have only a small effect on the reverse rate constant, koff'. Analysis of these data indicates that the rate laws governing the formation and dissociation of the [28 S:mRNA] complex cannot be deduced from the chemical equation. This observation suggests that there are "hidden intermediates' in the formation and dissociation of this complex. The implications of these observations are discussed in terms of a model for the interaction between the mitochondrial 28 S subunit and mRNAs.

  16. 13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit.

    PubMed

    Nishimura, Shinichi; Matsunaga, Shigeki; Yoshida, Minoru; Hirota, Hiroshi; Yokoyama, Shigeyuki; Fusetani, Nobuhiro

    2005-01-17

    13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens. In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated. [11-3H]-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate. Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit. In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation. Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues. These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.

  17. Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

    PubMed Central

    Lee, Sang-Won; Berger, Scott J.; Martinović, Suzana; Paša-Tolić, Ljiljana; Anderson, Gordon A.; Shen, Yufeng; Zhao, Rui; Smith, Richard D.

    2002-01-01

    Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by “mass locking” with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations. PMID:11983894

  18. Insights into remodeling events during eukaryotic large ribosomal subunit assembly provided by high resolution cryo-EM structures.

    PubMed

    Biedka, Stephanie; Wu, Shan; LaPeruta, Amber J; Gao, Ning; Woolford, John L

    2017-03-07

    Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.

  19. [Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method].

    PubMed

    Iusupov, M M; Spirin, A S

    1986-11-01

    A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.

  20. Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms.

    PubMed Central

    Telford, Maximilian J; Lockyer, Anne E; Cartwright-Finch, Chloë; Littlewood, D Timothy J

    2003-01-01

    The phylogenetic position of the phylum Platyhelminthes has been re-evaluated in the past decade by analysis of diverse molecular datasets. The consensus is that the Rhabditophora + Catenulida, which includes most of the flatworm taxa, are not primitively simple basal bilaterians but are related to coelomate phyla such as molluscs. The status of two other groups of acoelomate worms, Acoela and Nemertodermatida, is less clear. Although many characteristics unite these two groups, initial molecular phylogenetic studies placed the Nemertodermatida within the Rhabditophora, but placed the Acoela at the base of the Bilateria, distant from other flatworms. This contradiction resulted in scepticism about the basal position of acoels and led to calls for further data. We have sequenced large subunit ribosomal RNA genes from 13 rhabditophorans + catenulids, three acoels and one nemertodermatid, tripling the available data. Our analyses strongly support a basal position of both acoels and nemertodermatids. Alternative hypotheses are significantly less well supported by the data. We conclude that the Nemertodermatida and Acoela are basal bilaterians and, owing to their unique body plan and embryogenesis, should be recognized as a separate phylum, the Acoelomorpha. PMID:12803898

  1. eIF3j is located in the decoding center of the human 40S ribosomal subunit.

    PubMed

    Fraser, Christopher S; Berry, Katherine E; Hershey, John W B; Doudna, Jennifer A

    2007-06-22

    Protein synthesis in all cells begins with the ordered binding of the small ribosomal subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S subunit. Here we show by directed hydroxyl radical probing that the human eIF3 subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit, placing eIF3j directly in the ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.

  2. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    PubMed

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  3. Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

    PubMed

    Polkinghorne, Adam; Vaughan, Lloyd

    2011-01-01

    The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.

  4. Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining.

    PubMed

    Fringer, Jeanne M; Acker, Michael G; Fekete, Christie A; Lorsch, Jon R; Dever, Thomas E

    2007-03-01

    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.

  5. An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states.

    PubMed

    Blombach, Fabian; Launay, Helene; Zorraquino, Violeta; Swarts, Daan C; Cabrita, Lisa D; Benelli, Dario; Christodoulou, John; Londei, Paola; van der Oost, John

    2011-06-01

    HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.

  6. The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation

    PubMed Central

    Yonashiro, Ryo; Tahara, Erich B; Bengtson, Mario H; Khokhrina, Maria; Lorenz, Holger; Chen, Kai-Chun; Kigoshi-Tansho, Yu; Savas, Jeffrey N; Yates, John R; Kay, Steve A; Craig, Elizabeth A; Mogk, Axel; Bukau, Bernd; Joazeiro, Claudio AP

    2016-01-01

    Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension—the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation. DOI: http://dx.doi.org/10.7554/eLife.11794.001 PMID:26943317

  7. The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation.

    PubMed

    Yonashiro, Ryo; Tahara, Erich B; Bengtson, Mario H; Khokhrina, Maria; Lorenz, Holger; Chen, Kai-Chun; Kigoshi-Tansho, Yu; Savas, Jeffrey N; Yates, John R; Kay, Steve A; Craig, Elizabeth A; Mogk, Axel; Bukau, Bernd; Joazeiro, Claudio A P

    2016-03-04

    Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.

  8. Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

    PubMed

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I A; Matsumura, Takahiro; Tokoro, Masaharu

    2013-08-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

  9. The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates.

    PubMed

    Stoeck, T; Przybos, E; Dunthorn, M

    2014-05-01

    Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.

  10. Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

    PubMed Central

    Ivanova, Natalia; Pavlov, Michael Y.; Bouakaz, Elli; Ehrenberg, Måns; Schiavone, Lovisa Holmberg

    2005-01-01

    In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation. PMID:15972795

  11. Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

    PubMed

    Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

    2016-01-01

    The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

  12. DnaK-facilitated ribosome assembly in Escherichia coli revisited

    PubMed Central

    ALIX, JEAN-HERVÉ; NIERHAUS, KNUD H.

    2003-01-01

    Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30°C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37°C implicates a direct or indirect role for DnaK in this process. PMID:12810912

  13. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking

    SciTech Connect

    Uchiumi, T.; Wahba, A.J.; Traut, R.R.

    1987-08-01

    The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by diagonal (two-dimensional nonreducing-reducing) NaDodSO/sub 4//polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO/sub 4/ gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, PO. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO/sub 4//polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)/sub 2/ and (P2)/sub 2/ dimers, each of which is anchored to PO. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.

  14. Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit

    PubMed Central

    Li, Ningning; Chen, Yuling; Guo, Qiang; Zhang, Yixiao; Yuan, Yi; Ma, Chengying; Deng, Haiteng; Lei, Jianlin; Gao, Ning

    2013-01-01

    Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we characterized, both compositionally and structurally, a set of in vivo 50S subunit precursors (45S), isolated from a mutant bacterial strain. Our qualitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are dramatically underrepresented in the 45S particles. This protein spectrum shows interesting similarity to many qualitatively analyzed 50S precursors from different genetic background, indicating the presence of global rate-limiting steps in the late-stage assembly of 50S subunit. Our structural data reveal two major intermediate states for the 45S particles. Consistently, both states severally lack those proteins, but they also differ in the stability of the functional centers of the 50S subunit, demonstrating that they are translationally inactive. Detailed analysis indicates that the orientation of H38 accounts for the global conformational differences in these intermediate structures, and suggests that the reorientation of H38 to its native position is rate-limiting during the late-stage assembly. Especially, H38 plays an essential role in stabilizing the central protuberance, through the interaction with the 5S rRNA, and the correctly orientated H38 is likely a prerequisite for further maturation of the 50S subunit. PMID:23700310

  15. Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

    PubMed Central

    Dembowski, Jill A.; Ramesh, Madhumitha; McManus, C. Joel; Woolford, John L.

    2013-01-01

    Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3′ end of 5.8S rRNA and the 5′ end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing. PMID:24129494

  16. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation.

    PubMed

    Shao, Sichen; von der Malsburg, Karina; Hegde, Ramanujan S

    2013-06-06

    Quality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

  17. Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits.

    PubMed

    Doamekpor, Selom K; Lee, Joong-Won; Hepowit, Nathaniel L; Wu, Cheng; Charenton, Clement; Leonard, Marilyn; Bengtson, Mario H; Rajashankar, Kanagalaghatta R; Sachs, Matthew S; Lima, Christopher D; Joazeiro, Claudio A P

    2016-07-19

    The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1's substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1's intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD's role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

  18. Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

    PubMed Central

    Doamekpor, Selom K.; Lee, Joong-Won; Hepowit, Nathaniel L.; Wu, Cheng; Charenton, Clement; Leonard, Marilyn; Bengtson, Mario H.; Rajashankar, Kanagalaghatta R.; Sachs, Matthew S.; Lima, Christopher D.; Joazeiro, Claudio A. P.

    2016-01-01

    The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation. PMID:27385828

  19. Ribonuclease Sensitivity of Escherichia coli Ribosomes

    PubMed Central

    Santer, Melvin; Smith, Josephine R.

    1966-01-01

    Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099–1110. 1966.—The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10−4m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities. Images PMID:5332866

  20. DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation.

    PubMed

    Tummala, Hemanth; Walne, Amanda J; Williams, Mike; Bockett, Nicholas; Collopy, Laura; Cardoso, Shirleny; Ellison, Alicia; Wynn, Rob; Leblanc, Thierry; Fitzgibbon, Jude; Kelsell, David P; van Heel, David A; Payne, Elspeth; Plagnol, Vincent; Dokal, Inderjeet; Vulliamy, Tom

    2016-07-07

    A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.

  1. Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD.

    PubMed

    Kannangara, C G; Vothknecht, U C; Hansson, M; von Wettstein, D

    1997-03-18

    Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the

  2. Contributions of the N-terminal and C-terminal domains of initiation factor 3 towards its functions in the fidelity of initiation and anti-association of the ribosomal subunits.

    PubMed

    Ayyub, Shreya; Dobriyal, Divya; Varshney, Umesh

    2017-03-20

    Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of N- and C- terminal globular domains (NTD and CTD) joined by a linker region. IF3 is a ribosome anti-association factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli, and complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out ribosome anti-association function. Importantly, in the absence of NTD, bacterial growth is compromised, indicating a role for NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding, (a) initiation from non-AUG codons and, (b) initiation by initiator tRNAs lacking the highly conserved three consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.Importance Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit to the 30S pre-initiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

  3. Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p.

    PubMed Central

    Daugeron, M C; Kressler, D; Linder, P

    2001-01-01

    Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly. PMID:11565753

  4. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

    PubMed

    Shen, Peter S; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H; Cox, James; Cheng, Yifan; Lambowitz, Alan M; Weissman, Jonathan S; Brandman, Onn; Frost, Adam

    2015-01-02

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

  5. The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from its Structure in Crystals of 50S Ribosomal Subunits

    PubMed Central

    Shankar, Neelaabh; Kennedy, Scott D.; Chen, Gang; Krugh, Thomas R.; Turner, Douglas H.

    2014-01-01

    Internal loops play an important role in structure and folding of RNA and in RNA recognition by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5'1009CUAAG10133'3'1168GAAGC11645' and 5'998CUAAG10023'3'1157GAAGC11535' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex, 5'1GGCUAAGAC93'3'18CCGAAGCUG105' The internal loop forms a different structure in solution than in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson–Crick UA pair. PMID:17002278

  6. A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear small-subunit ribosomal DNA.

    PubMed

    Cook, Lyn G; Gullan, Penny J; Trueman, Holly E

    2002-10-01

    Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic.

  7. Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU).

    PubMed

    Pyziel, Anna M

    2014-03-01

    Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

  8. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  9. Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences.

    PubMed

    Gong, Ying-Chun; Yu, Yu-He; Zhu, Fei-Yun; Feng, Wei-Song

    2007-01-01

    To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.

  10. Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

    PubMed Central

    de la Cruz, J; Kressler, D; Rojo, M; Tollervey, D; Linder, P

    1998-01-01

    Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase. PMID:9769101

  11. The Structures of Antibiotics Bound to the E Site Region of the 50 S Ribosomal Subunit of Haloarcula marismortui: 13-Deoxytedanolide and Girodazole

    SciTech Connect

    Schroeder,S.; Blaha, G.; Tirado-Rives, J.; Steitz, T.; Moore, P.

    2007-01-01

    Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.

  12. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

    PubMed Central

    Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

    2015-01-01

    In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

  13. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

    PubMed Central

    Daugeron, M C; Linder, P

    1998-01-01

    Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. PMID:9582098

  14. Small subunit ribosomal DNA suggests that the xenophyophorean Syringammina corbicula is a foraminiferan.

    PubMed

    Pawlowski, Jan; Holzmann, Maria; Fahrni, Jose; Richardson, Susan L

    2003-01-01

    Xenophyophorea are giant deep-sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep-sea foraminiferan. Both species branch within a group of monothalamous (single-chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic-walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep-sea Foraminifera.

  15. Added resolution among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with complete small and large subunit nuclear ribosomal RNA genes.

    PubMed

    Waeschenbach, Andrea; Webster, Bonnie L; Bray, Rodney A; Littlewood, D T J

    2007-10-01

    The addition of large subunit ribosomal DNA (lsrDNA) to small subunit ribosomal DNA (ssrDNA) has been shown to add resolution to phylogenies at various taxonomic levels for a diversity of phyla. We added nearly complete lsrDNA (4057-4593bp) sequences to ssrDNA (1940-2228bp) for 26 ingroup and 3 outgroup taxa in an attempt to provide an improved ordinal phylogeny for the Cestoda. Ten lsrDNA and seven ssrDNA sequences were generated from new taxa and 13 existing partial lsrDNA sequences were sequenced to completion. The majority of phylogenetic signal in the combined analysis came from lsrDNA (69.6% of parsimonious informative sites, as opposed to 30.4% obtained from ssrDNA), resulting in almost identical topologies for lsrDNA and lsr+ssrDNA (pairwise symmetric distance=6) in model-based analyses. Topology testing found trees based on partial lsrDNA (domains D1-D3)+ssrDNA and complete lsr+ssrDNA to differ significantly; the addition of lsrDNA domains D4-D12 had a significant effect on topology. Overall nodal support was greatest in the combined analysis and weakest for ssrDNA only. Our molecular phylogenies differed significantly from those based on morphology alone. Acetabulate lineages form a monophyletic group, with the Tetraphyllidea being paraphyletic. Support for the combined data was high for the following topology: (Litobothriidea (Lecanicephalidea (Rhinebothrium/Rhodobothrium (Clistobothrium (Pachybothrium(Acanthobothrium Proteocephalidea) (Mesocestoididae, Nippotaeniidea, Cyclophyllidea, Tetrabothriidea)))))); all genus names refer to tetraphyllidean lineages. Although the interrelationships among the four most derived taxa remain uncertain, overall ambiguity of the acetabulate interrelationships was reduced. The Pseudophyllidea were recovered as polyphyletic, with support for a sister-group relationship between Diphyllobothriidae and Haplobothriidea. The monophyly of the Trypanorhyncha was recovered for the first time based on molecular data. The positions

  16. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

    PubMed

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří; Wilson, Daniel N

    2016-12-15

    Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

  17. Crystal Structures of EF-G-Ribosome Complexes Trapped in Intermediate States of Translocation

    SciTech Connect

    Zhou, Jie; Lancaster, Laura; Donohue, John Paul; Noller, Harry F.

    2013-11-12

    Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA–EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

  18. An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA.

    PubMed Central

    Nickrent, D L; Sargent, M L

    1991-01-01

    The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot. PMID:2014163

  19. Prevalent ciliate symbiosis on copepods: high genetic diversity and wide distribution detected using small subunit ribosomal RNA gene.

    PubMed

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.

  20. Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

    PubMed

    Hill, Kristina M; Stokes, Nancy A; Webb, Stephen C; Hine, P Mike; Kroeck, Marina A; Moore, James D; Morley, Margaret S; Reece, Kimberly S; Burreson, Eugene M; Carnegie, Ryan B

    2014-07-24

    The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

  1. Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

    PubMed Central

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally. PMID:23024768

  2. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

    NASA Astrophysics Data System (ADS)

    Hashem, Yaser; Des Georges, Amedee; Dhote, Vidya; Langlois, Robert; Liao, Hstau Y.; Grassucci, Robert A.; Pestova, Tatyana V.; Hellen, Christopher U. T.; Frank, Joachim

    2013-11-01

    Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

  3. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  4. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

  5. Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

    PubMed Central

    Bammert, Lukas; Jonas, Stefanie; Ungricht, Rosemarie; Kutay, Ulrike

    2016-01-01

    Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth. PMID:27599843

  6. Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata.

    PubMed

    Moreira, David; von der Heyden, Sophie; Bass, David; López-García, Purificación; Chao, Ema; Cavalier-Smith, Thomas

    2007-07-01

    Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of

  7. Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

    PubMed

    Li, Xiaojun; Sun, Qingan; Jiang, Cai; Yang, Kailu; Hung, Li-Wei; Zhang, Junjie; Sacchettini, James C

    2015-10-06

    The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a.

  8. A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments.

    PubMed

    Morgan, D G; Ménétret, J F; Radermacher, M; Neuhof, A; Akey, I V; Rapoport, T A; Akey, C W

    2000-08-11

    Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data. Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.

  9. Direct chemical probing of the conformation of the 3' functional domain of rabbit 18S rRNA in 40S subunits, 80S ribosomes and polyribosomes

    SciTech Connect

    Rubino, H.M.; Rairkar, A.; Lockard, R.E.

    1987-05-01

    Recent evidence suggests that the 3' minor domain of eukaryotic 18S rRNA, as in prokaryotes, is directly involved in protein biosynthesis. To determine regions of possible functional importance, they have probed the higher order structure of rabbit 18S rRNA in both 40S subunits and 80S ribosomes, as well as polyribosomes using the single-strand specific chemical probes dimethyl sulfate (DMS) and diethyl pyrocarbonate (DEPC) which react with unpaired guanosine and adenosine residues, respectively. The modified 18S rRNA was isolated from these particles and the resultant modified nucleotides identified on polyacrylamide sequencing gels upon either aniline-induced strand scission of /sup 32/P-end-labeled intact rRNA or by DNA primer extension using sequence specific deoxyoligomers with reverse transcriptase. Their results indicate a decreased reactivity of residue C-1692 in rabbit 18S rRNA (corresponding to C-1400 E. coli) within the putative tRNA contact site in polyribosomes as compared with 40S subunits and 80S ribosomes. They have also determined varying reactivities of a number of other residues within specific regions of the 3' functional domain when 40S, 80S, and polyribosomes are compared, which may be important for both subunit association as well as mRNA binding.

  10. Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin

    PubMed Central

    Chulluncuy, Roberto; Espiche, Carlos; Nakamoto, Jose Alberto; Fabbretti, Attilio; Milón, Pohl

    2016-01-01

    Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform. PMID:27983590

  11. Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.

    PubMed

    Chulluncuy, Roberto; Espiche, Carlos; Nakamoto, Jose Alberto; Fabbretti, Attilio; Milón, Pohl

    2016-12-13

    Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

  12. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-08

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  13. Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit

    PubMed Central

    Kurauskas, Vilius; Crublet, Elodie; Macek, Pavel; Kerfah, Rime; Gauto, Diego F.; Boisbouvier, Jérôme; Schanda, Paul

    2016-01-01

    Solid-state NMR spectroscopy allows the characterization of structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths only marginally increase. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus. PMID:27385633

  14. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  15. Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Kressler, D; de la Cruz, J; Rojo, M; Linder, P

    1997-01-01

    A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation. PMID:9372960

  16. UVB and gamma-radiation induce the expression of mRNAs encoding the ribosomal subunit L13A in rat keratinocytes.

    PubMed

    Shahmolky, N; Lefebvre, D L; Poon, R; Bai, Y; Sharma, M; Rosen, C F

    1999-09-01

    Ultraviolet B radiation produces an array of cellular perturbations in the skin. We isolated a keratinocyte cDNA encoding the rat 60S ribosomal subunit protein L13a following differential cDNA library screening with UVB-enriched probes. In contrast to the reported structure of liver L13a, the keratinocyte L13a cDNA contains a longer 3'-untranslated region. Northern blot analysis detected two L13a mRNA transcripts, approximately 800 bp and approximately 1.2 kb, in keratinocytes and a variety of rat tissues. Both L13a mRNA transcripts were induced by UVB irradiation, forskolin and gamma-irradiation. In contrast, no induction of L13a mRNA transcript levels was observed following exposure of keratinocytes to 12-O-tetradecanoylphorbol-13-acetate, serum and the DNA damage-inducing agents methyl methanesulfonate or 4-nitroquinoline-N-oxide. These observations suggest that increased expression of ribosomal subunit genes may be a molecular component of the keratinocyte response to UVB in particular and not part of a nonspecific response to DNA damage.

  17. Chlamydophila pneumoniae HflX belongs to an uncharacterized family of conserved GTPases and associates with the Escherichia coli 50S large ribosomal subunit.

    PubMed

    Polkinghorne, Adam; Ziegler, Urs; González-Hernández, Yanela; Pospischil, Andreas; Timms, Peter; Vaughan, Lloyd

    2008-11-01

    Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [gamma-(32)P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

  18. Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

    PubMed

    Ermolenko, Dmitri N; Cornish, Peter V; Ha, Taekjip; Noller, Harry F

    2013-02-01

    In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

  19. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    NASA Technical Reports Server (NTRS)

    Winker, S.; Woese, C. R.

    1991-01-01

    The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

  20. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    SciTech Connect

    Winker, S.; Woese, C.R.

    1994-11-01

    The number of small subunit rRNA sequences is not great enough that the three domains Archaea, Bacteria, and Eucarya (Woese, et al., 1990) can be reliably defined in terms of their sequence ``signatures.`` Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterized and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic composition at approximately fifteen positions in the small subunit rRNA molecule.

  1. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit.

    PubMed

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G; Maher, Kathryn N; Lorsch, Jon R; Dever, Thomas E

    2009-02-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

  2. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

    PubMed

    Golovin, A V; Khayrullina, G A; Kraal, B; Kopylov, Capital A Cyrillic М

    2012-10-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

  3. RibAlign: a software tool and database for eubacterial phylogeny based on concatenated ribosomal protein subunits

    PubMed Central

    Teeling, Hanno; Gloeckner, Frank Oliver

    2006-01-01

    Background Until today, analysis of 16S ribosomal RNA (rRNA) sequences has been the de-facto gold standard for the assessment of phylogenetic relationships among prokaryotes. However, the branching order of the individual phlya is not well-resolved in 16S rRNA-based trees. In search of an improvement, new phylogenetic methods have been developed alongside with the growing availability of complete genome sequences. Unfortunately, only a few genes in prokaryotic genomes qualify as universal phylogenetic markers and almost all of them have a lower information content than the 16S rRNA gene. Therefore, emphasis has been placed on methods that are based on multiple genes or even entire genomes. The concatenation of ribosomal protein sequences is one method which has been ascribed an improved resolution. Since there is neither a comprehensive database for ribosomal protein sequences nor a tool that assists in sequence retrieval and generation of respective input files for phylogenetic reconstruction programs, RibAlign has been developed to fill this gap. Results RibAlign serves two purposes: First, it provides a fast and scalable database that has been specifically adapted to eubacterial ribosomal protein sequences and second, it provides sophisticated import and export capabilities. This includes semi-automatic extraction of ribosomal protein sequences from whole-genome GenBank and FASTA files as well as exporting aligned, concatenated and filtered sequence files that can directly be used in conjunction with the PHYLIP and MrBayes phylogenetic reconstruction programs. Conclusion Up to now, phylogeny based on concatenated ribosomal protein sequences is hampered by the limited set of sequenced genomes and high computational requirements. However, hundreds of full and draft genome sequencing projects are on the way, and advances in cluster-computing and algorithms make phylogenetic reconstructions feasible even with large alignments of concatenated marker genes. Rib

  4. RIBOSOME-MEMBRANE INTERACTION

    PubMed Central

    Adelman, M. R.; Sabatini, David D.; Blobel, Günter

    1973-01-01

    In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear. PMID:4682341

  5. Atomic mutagenesis at the ribosomal decoding site.

    PubMed

    Schrode, Pius; Huter, Paul; Clementi, Nina; Erlacher, Matthias

    2017-01-02

    Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an in vitro reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding.

  6. Atomic mutagenesis at the ribosomal decoding site

    PubMed Central

    Schrode, Pius; Huter, Paul; Clementi, Nina; Erlacher, Matthias

    2017-01-01

    ABSTRACT Ribosomal decoding is an essential process in every living cell. During protein synthesis the 30S ribosomal subunit needs to accomplish binding and accurate decoding of mRNAs. From mutational studies and high-resolution crystal structures nucleotides G530, A1492 and A1493 of the 16S rRNA came into focus as important elements for the decoding process. Recent crystallographic data challenged the so far accepted model for the decoding mechanism. To biochemically investigate decoding in greater detail we applied an in vitro reconstitution approach to modulate single chemical groups at A1492 and A1493. The modified ribosomes were subsequently tested for their ability to efficiently decode the mRNA. Unexpectedly, the ribosome was rather tolerant toward modifications of single groups either at the base or at the sugar moiety in terms of translation activity. Concerning translation fidelity, the elimination of single chemical groups involved in a hydrogen bonding network between the tRNA, mRNA and rRNA did not change the accuracy of the ribosome. These results indicate that the contribution of those chemical groups and the formed hydrogen bonds are not crucial for ribosomal decoding. PMID:27841727

  7. A recent intermezzo at the Ribosome Club.

    PubMed

    Pavlov, Michael Y; Liljas, Anders; Ehrenberg, Måns

    2017-03-19

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg(2+) ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account.This article is part of the themed issue 'Perspectives on the ribosome'.

  8. Discrimination between Gyrodactylus salaris, G. derjavini and G. truttae (Platyhelminthes: Monogenea) using restriction fragment length polymorphisms and an oligonucleotide probe within the small subunit ribosomal RNA gene.

    PubMed

    Cunningham, C O; McGillivray, D M; MacKenzie, K; Melvin, W T

    1995-07-01

    The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region.

  9. Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences.

    PubMed

    Carreno, R A; Martin, D S; Barta, J R

    1999-11-01

    The phylogenetic placement of gregarine parasites (Apicomplexa: Gregarinasina) within the Apicomplexa was derived by comparison of small-subunit ribosomal RNA gene sequences. Gregarine sequences were obtained from Gregarina niphandrodes Clopton, Percival, and Janovy, 1991, and Monocystis agilis Stein, 1848 (Eugregarinorida Léger 1900), as well as from Ophriocystis elektroscirrha McLaughlin and Myers, 1970 (Neogregarinorida Grassé 1953). The sequences were aligned with several other gregarine and apicomplexan sequences from GenBank and the resulting data matrix analyzed by parsimony and maximum-likelihood methods. The gregarines form a monophyletic clade that is a sister group to Cryptosporidium spp. The gregarine/ Cryptosporidium clade is separate from the other major apicomplexan clade containing the coccidia, adeleids, piroplasms, and haemosporinids. The trees indicate that the genus Cryptosporidium has a closer phylogenetic affinity with the gregarines than with the coccidia. These results do not support the present classification of the Cryptosporidiidae in the suborder Eimerioirina Léger, 1911.

  10. Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

    PubMed Central

    Gupta, Aditya K.; Boekhout, Teun; Theelen, Bart; Summerbell, Richard; Batra, Roma

    2004-01-01

    Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains. PMID:15365020

  11. The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species.

    PubMed

    Vrba, Vladimir; Poplstein, Martin; Pakandl, Michal

    2011-12-29

    Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

  12. The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1

    PubMed Central

    Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Jarolim, Stefanie; Simon-Nobbe, Birgit; Hager, Matthias; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

    2009-01-01

    Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging. PMID:20157544

  13. The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

    PubMed

    Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

    2009-07-13

    Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

  14. Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of the sequencing effort, rarefaction analysis of the ribosomal small sub-unit (SSU/16S/18...

  15. Phylogeny of organisms investigated by the base-pair changes in the stem regions of small and large ribosomal subunit RNAs.

    PubMed

    Otsuka, J; Terai, G; Nakano, T

    1999-02-01

    In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs). The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts. Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs. The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation. This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth.

  16. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  17. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  18. Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

    PubMed

    Du, Zhi-Qiang; Li, Xin; Wang, Jian-Ying

    2016-08-01

    Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein.

  19. A recent intermezzo at the Ribosome Club

    PubMed Central

    Pavlov, Michael Y.; Liljas, Anders

    2017-01-01

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account. This article is part of the themed issue ‘Perspectives on the ribosome’. PMID:28138071

  20. RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits.

    PubMed Central

    Fabian, G R; Hopper, A K

    1987-01-01

    The Saccharomyces cerevisiae mutant ts351 had been shown to affect processing of 27S pre-rRNA to mature 25S and 5.8S rRNAs (C. Andrew, A. K. Hopper, and B. D. Hall, Mol. Gen. Genet. 144:29-37, 1976). We showed that this strain contains two mutations leading to temperature-sensitive lethality. The rRNA-processing defect, however, is a result of only one of the two mutations. We designated the lesion responsible for the rRNA-processing defect rrp1 and showed that it is located on the right arm of chromosome IV either allelic to or tightly linked to mak21. This rrp1 lesion also results in hypersensitivity to aminoglycoside antibiotics and a reduced 25S/18S rRNA ratio at semipermissive temperatures. We cloned the RRP1 gene and provide evidence that it encodes a moderately abundant mRNA which is in lower abundance and larger than most mRNAs encoding ribosomal proteins. Images PMID:3549696

  1. An Introduction to the Structure and Function of the Ribosome.

    PubMed

    Dunkle, Jack A; Cate, Jamie H D

    2013-02-01

    E. coli continues to serve as a key model for the structure and function of the ribosome, structures of ribosome from other organisms and domains of life have also greatly contributed to our knowledge of protein synthesis. Many structural models of the ribosome in a number of steps of the protein synthesis cycle have been solved by cryo-electron microscopy (cryo-EM) and x-ray crystallography. This chapter introduces the structure and dynamics of the ribosome based on these structures and ends with a brief discussion of the many questions that the structures leave unanswered. Protein synthesis is a multistep process, and the structural features of the ribosome along with the large number of cofactors reflect the complexity of translation. Numerous protein factors in addition to the ribosome contribute to translation in bacteria during the steps of initiation, elongation, termination, and recycling. These protein factors make intimate contacts to key regions of the ribosome, and this aspect is discussed in the chapter in light of our present understanding of the structure and function of the ribosome. The intact ribosome contains three binding sites for substrate tRNAs that are termed as the aminoacyl-tRNA site (A site), peptidyl-tRNA site (P site), and exit-tRNA site (E site). These three binding sites span the interface between the 30S and 50S subunits. The central activity of the ribosome is catalysis of peptide bond formation. The region of the ribosome responsible for catalyzing the reaction is called the peptidyl transferase center (PTC).

  2. The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes.

    PubMed

    Zhao, Huayan; Lü, Shiyou; Li, Ruixi; Chen, Tao; Zhang, Huoming; Cui, Peng; Ding, Feng; Liu, Pei; Wang, Guangchao; Xia, Yiji; Running, Mark P; Xiong, Liming

    2015-11-01

    The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

  3. Prevalence, Genetic Characterization, and 18S Small Subunit Ribosomal RNA Diversity of Trypanosoma rangeli in Triatomine and Mammal Hosts in Endemic Areas for Chagas Disease in Ecuador

    PubMed Central

    Ocaña-Mayorga, Sofia; Aguirre-Villacis, Fernanda; Pinto, C. Miguel; Vallejo, Gustavo A.

    2015-01-01

    Abstract Trypanosoma rangeli is a nonpathogenic parasite for humans; however, its medical importance relies in its similarity and overlapping distribution with Trypanosoma cruzi, causal agent of Chagas disease in the Americas. The genetic diversity of T. rangeli and its association with host species (triatomines and mammals) has been identified along Central and the South America; however, it has not included data of isolates from Ecuador. This study reports infection with T. rangeli in 18 genera of mammal hosts and five species of triatomines in three environments (domestic, peridomestic, and sylvatic). Higher infection rates were found in the sylvatic environment, in close association with Rhodnius ecuadoriensis. The results of this study extend the range of hosts infected with this parasite and the geographic range of the T. rangeli genotype KP1(−)/lineage C in South America. It was not possible to detect variation on T. rangeli from the central coastal region and southern Ecuador with the analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, even though these areas are ecologically different and a phenotypic subdivision of R. ecuadoriensis has been found. R. ecuadoriensis is considered one of the most important vectors for Chagas disease transmission in Ecuador due to its wide distribution and adaptability to diverse environments. An extensive knowledge of the trypanosomes circulating in this species of triatomine, and associated mammal hosts, is important for delineating transmission dynamics and preventive measures in the endemic areas of Ecuador and Northern Peru. PMID:26645579

  4. The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript1[OPEN

    PubMed Central

    Sanglard, Lilian Vincis Pereira; Bussell, John D.; Howell, Katharine A.

    2017-01-01

    We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves. PMID:28011633

  5. Genetic variation of Gongylonema pulchrum from wild animals and cattle in Japan based on ribosomal RNA and mitochondrial cytochrome c oxidase subunit I genes.

    PubMed

    Makouloutou, P; Setsuda, A; Yokoyama, M; Tsuji, T; Saita, E; Torii, H; Kaneshiro, Y; Sasaki, M; Maeda, K; Une, Y; Hasegawa, H; Sato, H

    2013-09-01

    The gullet worm (Gongylonema pulchrum) has been recorded from a variety of mammals worldwide, including monkeys and humans. Due to its wide host range, it has been suggested that the worm may be transmitted locally to any mammalian host by chance. To investigate this notion, the ribosomal RNA gene (rDNA), mainly regions of the internal transcribed spacers (ITS) 1 and 2, and a cytochrome c oxidase subunit I (COI) region of mitochondrial DNA of G. pulchrum were characterized using parasites from the following hosts located in Japan: cattle, sika deer, wild boars, Japanese macaques, a feral Reeves's muntjac and captive squirrel monkeys. The rDNA nucleotide sequences of G. pulchrum were generally well conserved regardless of their host origin. However, a few insertions/deletions of nucleotides along with a few base substitutions in the ITS1 and ITS2 regions were observed in G. pulchrum from sika deer, wild boars and Japanese macaques, and those differed from G. pulchrum in cattle, the feral Reeves's muntjac and captive squirrel monkeys. The COI sequences of G. pulchrum were further divided into multiple haplotypes and two groups of haplotypes, i.e. those from a majority of sika deer, wild boars and Japanese macaques and those from cattle and zoo animals, were clearly differentiated. Our findings indicate that domestic and sylvatic transmission cycles of the gullet worm are currently present, at least in Japan.

  6. Prevalence, Genetic Characterization, and 18S Small Subunit Ribosomal RNA Diversity of Trypanosoma rangeli in Triatomine and Mammal Hosts in Endemic Areas for Chagas Disease in Ecuador.

    PubMed

    Ocaña-Mayorga, Sofia; Aguirre-Villacis, Fernanda; Pinto, C Miguel; Vallejo, Gustavo A; Grijalva, Mario J

    2015-12-01

    Trypanosoma rangeli is a nonpathogenic parasite for humans; however, its medical importance relies in its similarity and overlapping distribution with Trypanosoma cruzi, causal agent of Chagas disease in the Americas. The genetic diversity of T. rangeli and its association with host species (triatomines and mammals) has been identified along Central and the South America; however, it has not included data of isolates from Ecuador. This study reports infection with T. rangeli in 18 genera of mammal hosts and five species of triatomines in three environments (domestic, peridomestic, and sylvatic). Higher infection rates were found in the sylvatic environment, in close association with Rhodnius ecuadoriensis. The results of this study extend the range of hosts infected with this parasite and the geographic range of the T. rangeli genotype KP1(-)/lineage C in South America. It was not possible to detect variation on T. rangeli from the central coastal region and southern Ecuador with the analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, even though these areas are ecologically different and a phenotypic subdivision of R. ecuadoriensis has been found. R. ecuadoriensis is considered one of the most important vectors for Chagas disease transmission in Ecuador due to its wide distribution and adaptability to diverse environments. An extensive knowledge of the trypanosomes circulating in this species of triatomine, and associated mammal hosts, is important for delineating transmission dynamics and preventive measures in the endemic areas of Ecuador and Northern Peru.

  7. Binding of Dihydrostreptomycin to Escherichia coli Ribosomes: Kinetics of the Reaction

    PubMed Central

    Chang, F. N.; Flaks, Joel G.

    1972-01-01

    Investigations were carried out on the binding of dihydrostreptomycin to purified (and reassociated) 70S ribosomes and 30S subunits from streptomycin-susceptible strains, and the results were compared with those of similar studies with native (run-off) 70S ribosomes. At 0 C, only a small fraction of purified 70S ribosomes and 30S sub-units bound 1 molecule of the antibiotic tightly, and at a rate comparable to the binding occurring with native 70S ribosomes. At temperatures of 10 C and above, there was a temperature-dependent increase in the extent of antibiotic binding to purified 70S and 30S particles up to a maximum of 1 molecule/ribosomal particle, but the kinetics of binding was slow in comparison to that taking place at 0 C. These and other results suggest that a major fraction of 30S subunits and purified (or reassociated) 70S ribosomes are inactive in binding the antibiotic. This has been localized to an instability of the free 30S subunit, which in solution at 0 C has a half-life of 5 hr or less. Inactive 30S or 70S particles could be thermally activated, with the latter being identical in their streptomycin-binding properties to native 70S ribosomes. The activation kinetics were slow in comparison to the binding kinetics for the antibiotic and were indicative of a conformational change in ribosomal structure. There thus appears to be a reversible transition between active and inactive forms of the ribosomal particles for streptomycin binding, but additional binding sites for the antibiotic are not created by the transitions. The active form of the 30S subunit can be stabilized in the presence of polyuridylic acid, but much more effectively by association with the 50S subunit to form a 70S ribosome. The kinetics of dihydrostreptomycin binding were studied in both directions of the reaction, and the reaction in the direction of binding was found to be several orders of magnitude faster than that of the reverse, or debinding, direction. The kinetics of the

  8. Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.

    PubMed

    Fu, Ziao; Kaledhonkar, Sandip; Borg, Anneli; Sun, Ming; Chen, Bo; Grassucci, Robert A; Ehrenberg, Måns; Frank, Joachim

    2016-12-06

    Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

  9. The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes

    PubMed Central

    Wegrecki, Marcin; Rodríguez-Galán, Olga; de la Cruz, Jesús; Bravo, Jeronimo

    2015-01-01

    Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the β-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles. PMID:26476442

  10. The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes.

    PubMed

    Wegrecki, Marcin; Rodríguez-Galán, Olga; de la Cruz, Jesús; Bravo, Jeronimo

    2015-12-15

    Ribosome biogenesis is one of the most essential pathways in eukaryotes although it is still not fully characterized. Given the importance of this process in proliferating cells, it is obvious that understanding the macromolecular details of the interactions that take place between the assembly factors, ribosomal proteins and nascent pre-rRNAs is essentially required for the development of new non-genotoxic treatments for cancer. Herein, we have studied the association between the WD40-repeat domains of Erb1 and Ytm1 proteins. These are essential factors for the biogenesis of 60S ribosomal subunits in eukaryotes that form a heterotrimeric complex together with the also essential Nop7 protein. We provide the crystal structure of a dimer formed by the C-terminal part of Erb1 and Ytm1 from Chaetomium thermophilum at 2.1 Å resolution. Using a multidisciplinary approach we show that the β-propeller domains of these proteins interact in a novel manner that leads to a high-affinity binding. We prove that a point mutation within the interface of the complex impairs the interaction between the two proteins and negatively affects growth and ribosome production in yeast. Our study suggests insights into the association of the Erb1-Ytm1 dimer with pre-ribosomal particles.

  11. Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

    PubMed

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-03-11

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.

  12. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit.

    PubMed

    Garner, Cherilyn D; Starr, Jennifer K; McDonough, Patrick L; Altier, Craig

    2010-06-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

  13. Using Small Subunit Ribosomal RNA to Follow Dark Incorporation of 14C-bicarbonate by Bacteria and Archaea in Sandy Sediment

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Musat, N.; Kuypers, M. M.

    2007-12-01

    Small subunit ribosomal RNA (SSU rRNA) and the genes encoding it have become the basis of modern microbial phylogeny, and of numerous methods for characterizing the composition of bacterial, archaeal, and even eukaryotic communities as they occur in nature. A limitation of this approach has been that phylogeny alone is not a reliable guide to physiology, particularly for groups with no close relatives in culture. We have been developing ways of using the SSU rRNA molecule itself to identify and (eventually) quantify the carbon sources incorporated by particular phylogenetic groups. This can be done by taking advantage of natural variations in carbon isotopic composition among growth substrates, or by following incorporation of 13C- or 14C-labeled compounds. 14C has the advantage that natural background levels are negligible. In the present study, our goal is to identify species responsible for non-photosynthetic CO2 incorporation in sandy sediments of the German Wadden Sea. Sediment cores collected from the Janssand sand flats were percolated with 14C-bicarbonate at in situ temperature for 36-38h in the dark, total RNA isolated, and domain-specific oligonucleotide probes used to capture bacterial and archaeal SSU rRNA. Total and/or captured RNA was separated by denaturing polyacrylamide gel electrophoresis, and 14C detected by phosphor imager, autoradiography, or beta imager. Detection was fastest and most sensitive with the beta imager. Both Bacteria and Archaea had incorporated label, suggesting both groups may harbor non-photosynthetic autotrophs. The next step will be to use more specific capture probes. We are currently working to separate the captured domain-specific SSU rRNA on non-denaturing gels, with detection by the high-resolution mode of the beta imager, so that individual species incorporating label can be identified by RT-PCR and sequencing of labeled bands.

  14. RBF1, a Plant Homolog of the Bacterial Ribosome-Binding Factor RbfA, Acts in Processing of the Chloroplast 16S Ribosomal RNA1[W

    PubMed Central

    Fristedt, Rikard; Scharff, Lars B.; Clarke, Cornelia A.; Wang, Qin; Lin, Chentao; Merchant, Sabeeha S.; Bock, Ralph

    2014-01-01

    Plastids (chloroplasts) possess 70S ribosomes that are very similar in structure and function to the ribosomes of their bacterial ancestors. While most components of the bacterial ribosome (ribosomal RNAs [rRNAs] and ribosomal proteins) are well conserved in the plastid ribosome, little is known about the factors mediating the biogenesis of plastid ribosomes. Here, we have investigated a putative homolog of the bacterial RbfA (for ribosome-binding factor A) protein that was identified as a cold-shock protein and an auxiliary factor acting in the 5′ maturation of the 16S rRNA. The unicellular green alga Chlamydomonas reinhardtii and the vascular plant Arabidopsis (Arabidopsis thaliana) both encode a single RbfA-like protein in their nuclear genomes. By generating specific antibodies against this protein, we show that the plant RbfA-like protein functions exclusively in the plastid, where it is associated with thylakoid membranes. Analysis of mutants for the corresponding gene (termed RBF1) reveals that the gene function is essential for photoautotrophic growth. Weak mutant alleles display reduced levels of plastid ribosomes, a specific depletion in 30S ribosomal subunits, and reduced activity of plastid protein biosynthesis. Our data suggest that, while the function in ribosome maturation and 16S rRNA 5′ end processing is conserved, the RBF1 protein has assumed an additional role in 3′ end processing. Together with the apparent absence of a homologous protein from plant mitochondria, our findings illustrate that the assembly process of the 70S ribosome is not strictly conserved and has undergone some modifications during organelle evolution. PMID:24214533

  15. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  16. Ribosomal protein L3 functions as a ‘rocker switch’ to aid in coordinating of large subunit-associated functions in eukaryotes and Archaea

    PubMed Central

    Meskauskas, Arturas

    2008-01-01

    Although ribosomal RNAs (rRNAs) comprise the bulk of the ribosome and carry out its main functions, ribosomal proteins also appear to play important structural and functional roles. Many ribosomal proteins contain long, nonglobular domains that extend deep into the rRNA cores. In eukaryotes and Archaea, ribosomal protein L3 contains two such extended domains tethered to a common globular hub, thus providing an excellent model to address basic questions relating to ribosomal protein structure/function relationships. Previous work in our laboratory identified the central ‘W-finger’ extension of yeast L3 in helping to coordinate ribosomal functions. New studies on the ‘N-terminal’ extension in yeast suggest that it works with the W-finger to coordinate opening and closing of the corridor through which the 3′ end of aa-tRNA moves during the process of accommodation. Additionally, the effect of one of the L3 N-terminal extension mutants on the interaction between C75 of the aa-tRNA and G2921 (Escherichia coli G2553) of 25S rRNA provides the first evidence of the effect of a ribosomal protein on aa-tRNA positioning and peptidyltransfer, possibly through the induced fit model. A model is presented describing how all three domains of L3 may function together as a ‘rocker switch’ to coordinate the stepwise processes of translation elongation. PMID:18832371

  17. Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

    PubMed Central

    Lempereur, L; Nicoloso, M; Riehl, N; Ehresmann, C; Ehresmann, B; Bachellerie, J P

    1985-01-01

    The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized. Images PMID:2417197

  18. Concentric-Flow Electrokinetic Injector Enables Serial Crystallography of Ribosome and Photosystem-II

    PubMed Central

    Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan; Dao, E. Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S.; Young, Iris D.; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S.; Koglin, Jason E.; Boutet, Sébastien; Junco, Elia A.; Hayes, Brandon; Bogan, Michael J.; Hampton, Christina Y.; Puglisi, Elisabetta V.; Sauter, Nicholas K.; Stan, Claudiu A.; Zouni, Athina; Yano, Junko; Yachandra, Vittal K.; Soltis, S. Michael; Puglisi, Joseph D.; DeMirci, Hasan

    2016-01-01

    In this work, a concentric-flow electrokinetic injector delivered microcrystals of Geobacillus stearothermophilus thermolysin (2.2 Å structure), Thermosynechococcus elongatus photosystem II (< 3 Å diffraction) and Thermus thermophilus small ribosomal subunit (3.4 Å structure). The first ambient-temperature X-ray crystal structure of the 30S subunit bound to the antibiotic paromomycin was obtained in its native mother liquor. Compared to previous cryo-cooled structures, this new structure showed that paromomycin binds to the decoding center in a different conformation. PMID:26619013

  19. Discovery of a small molecule that inhibits bacterial ribosome biogenesis

    PubMed Central

    Stokes, Jonathan M; Davis, Joseph H; Mangat, Chand S; Williamson, James R; Brown, Eric D

    2014-01-01

    While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15°C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly. DOI: http://dx.doi.org/10.7554/eLife.03574.001 PMID:25233066

  20. Steric interactions lead to collective tilting motion in the ribosome during mRNA–tRNA translocation

    PubMed Central

    Nguyen, Kien; Whitford, Paul C.

    2016-01-01

    Translocation of mRNA and tRNA through the ribosome is associated with large-scale rearrangements of the head domain in the 30S ribosomal subunit. To elucidate the relationship between 30S head dynamics and mRNA–tRNA displacement, we apply molecular dynamics simulations using an all-atom structure-based model. Here we provide a statistical analysis of 250 spontaneous transitions between the A/P–P/E and P/P–E/E ensembles. Consistent with structural studies, the ribosome samples a chimeric ap/P–pe/E intermediate, where the 30S head is rotated ∼18°. It then transiently populates a previously unreported intermediate ensemble, which is characterized by a ∼10° tilt of the head. To identify the origins of head tilting, we analyse 781 additional simulations in which specific steric features are perturbed. These calculations show that head tilting may be attributed to specific steric interactions between tRNA and the 30S subunit (PE loop and protein S13). Taken together, this study demonstrates how molecular structure can give rise to large-scale collective rearrangements. PMID:26838673

  1. Structural basis for the rescue of stalled ribosomes: structure of YaeJ bound to the ribosome.

    PubMed

    Gagnon, Matthieu G; Seetharaman, Sai V; Bulkley, David; Steitz, Thomas A

    2012-03-16

    In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA(i)(fMet) and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.

  2. Structural Basis for the Rescue of Stalled Ribosomes: Structure of YaeJ Bound to the Ribosome

    SciTech Connect

    Gagnon, Matthieu G.; Seetharaman, Sai V.; Bulkley, David; Steitz, Thomas A.

    2012-06-19

    In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA{sub i}{sup fMet} and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.

  3. Structural Basis for Translation Termination on the 70S Ribosome

    SciTech Connect

    Laurberg, M.; Asahara, H.; Korostelev, A.; Zhu, J.; Trakhanov, S.; Noller, H.F.

    2009-05-20

    At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 {angstrom} resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.

  4. Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes.

    PubMed

    Wickramasinghe, Susiji; Yatawara, Lalani; Rajapakse, R P V J; Agatsuma, Takeshi

    2009-06-01

    Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.

  5. The interaction between human initiation factor eIF3 subunit c and heat-shock protein 90: a necessary factor for translation mediated by the hepatitis C virus internal ribosome entry site.

    PubMed

    Ujino, Saneyuki; Nishitsuji, Hironori; Sugiyama, Ryuichi; Suzuki, Hitoshi; Hishiki, Takayuki; Sugiyama, Kazuo; Shimotohno, Kunitada; Takaku, Hiroshi

    2012-01-01

    Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives translation by directly recruiting the 40S ribosomal subunits that bind to eukaryotic initiation factor 3 (eIF3). Our data indicate that Hsp90 binds indirectly to eIF3 subunit c by interacting with it through the HCV IRES RNA, and the functional consequence of this Hsp90-eIF3c-HCV-IRES RNA interaction is the prevention of ubiquitination and the proteasome-dependent degradation of eIF3c. Hsp90 activity interference by Hsp90 inhibitors appears to be the result of the dissociation of eIF3c from Hsp90 in the presence of HCV IRES RNA and the resultant induction of the degradation of the free forms of eIF3c. Moreover, the interaction between Hsp90 and eIF3c is dependent on HCV IRES RNA binding. Furthermore, we demonstrate, by knockdown of eIF3c, that the silencing of eIF3c results in inhibitory effects on translation of HCV-derived RNA but does not affect cap-dependent translation. These results indicate that the interaction between Hsp90 and eIF3c may play an important role in HCV IRES-mediated translation.

  6. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

  7. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

    PubMed Central

    Alix, J H; Guérin, M F

    1993-01-01

    To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature. PMID:8105482

  8. Role of Protein L25 and Its Contact with Protein L16 in Maintaining the Active State of Escherichia coli Ribosomes in vivo.

    PubMed

    Anikaev, A Y; Isaev, A B; Korobeinikova, A V; Garber, M B; Gongadze, G M

    2016-01-01

    A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.

  9. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. coli

    PubMed Central

    Golovin, A.V.; Khayrullina, G.A.; Kraal, B.; Kopylov, А.М.

    2012-01-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA–protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA–protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV–induced RNA–protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA– protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA – protein cross–link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA–protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit. PMID:23346381

  10. Preparation and proteomic analysis of chloroplast ribosomes.

    PubMed

    Yamaguchi, Kenichi

    2011-01-01

    Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.

  11. Evolutionarily Conserved Function of RRP36 in Early Cleavages of the Pre-rRNA and Production of the 40S Ribosomal Subunit ▿ †

    PubMed Central

    Gérus, Marie; Bonnart, Chrystelle; Caizergues-Ferrer, Michèle; Henry, Yves; Henras, Anthony K.

    2010-01-01

    Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes. PMID:20038530

  12. Massively Convergent Evolution for Ribosomal Protein Gene Content in Plastid and Mitochondrial Genomes

    PubMed Central

    Maier, Uwe-G; Zauner, Stefan; Woehle, Christian; Bolte, Kathrin; Hempel, Franziska; Allen, John F.; Martin, William F.

    2013-01-01

    Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force. PMID:24259312

  13. The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

    PubMed

    Chiaberge, S; Cassarino, E; Mangiarotti, G

    1998-10-16

    AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

  14. Discovery of a Katablepharis sp. in the Columbia River estuary that is abundant during the spring and bears a unique large ribosomal subunit sequence element

    PubMed Central

    Kahn, Peter; Herfort, Lydie; Peterson, Tawnya D; Zuber, Peter

    2014-01-01

    Heterotrophic protists play significant roles in pelagic food webs as bacterivorous and herbivorous consumers. However, heterotrophic protists—unlike autotrophic ones—are often difficult to track since they tend to lack features such as photosynthetic pigments that allow for remote sensing or for bulk characterization. Difficulty in the identification of heterotrophic protists has often resulted in lumping them into broad groups, but there is a strong need to develop methods that increase the spatial and temporal resolution of observations applied to particular organisms in order to discover the drivers of population structure and ecological function. In surveys of small subunit rRNA, gene (SSU) sequences of microbial eukaryotes from the Columbia River to the Pacific Ocean, the heterotrophic flagellate Katablepharis sp. were found to dominate protist assemblages (including autotrophic and heterotrophic fractions) in the spring, prior to the freshet. We discovered a 332 base pair unique sequence element (USE) insertion in the large subunit rRNA gene (28S) that is not present in other katablepharids or in any other eukaryote. Using this USE, we were able to detect Katablepharis within mixed assemblages in river, estuarine, and oceanic samples and determine spatial and temporal patterns in absolute abundance through quantitative PCR and fluorescence in situ hybridization. Given their high abundance and repeatable temporal patterns of occurrence, we hypothesize that the Columbia River Estuary Katablepharis (Katablepharis CRE) plays an important role in estuarine biogeochemical and ecosystem function. PMID:25168204

  15. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo

    PubMed Central

    Cheung, Yuen-Nei; Maag, David; Mitchell, Sarah F.; Fekete, Christie A.; Algire, Mikkel A.; Takacs, Julie E.; Shirokikh, Nikolay; Pestova, Tatyana; Lorsch, Jon R.; Hinnebusch, Alan G.

    2007-01-01

    Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2•GTP•Met-tRNAiMet ternary complex (TC) and subsequent Pi release from eIF2•GDP•Pi. It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking Pi release at non-AUG codons. We show that Sui− mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui− mutation ISQLG93–97ASQAA (abbreviated 93–97) accelerates eIF1 dissociation and Pi release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui− mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd− mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection. PMID:17504939

  16. Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

    PubMed

    Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

    2011-10-01

    The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

  17. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  18. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    SciTech Connect

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  19. Ribosome engineering to promote new crystal forms

    SciTech Connect

    Selmer, Maria; Gao, Yong-Gui; Weixlbaumer, Albert; Ramakrishnan, V.

    2012-05-01

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  20. Ribosomal Database Project II

    DOE Data Explorer

    The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences. As of March 2008, RDP Release 10 is available and currently (August 2009) contains 1,074,075 aligned 16S rRNA sequences. Data that can be downloaded include zipped GenBank and FASTA alignment files, a histogram (in Excel) of the number of RDP sequences spanning each base position, data in the Functional Gene Pipeline Repository, and various user submitted data. The RDP-II website also provides numerous analysis tools.[From the RDP-II home page at http://rdp.cme.msu.edu/index.jsp

  1. Biphasic character of ribosomal translocation and non-Michaelis-Menten kinetics of translation.

    PubMed

    Xie, Ping

    2014-12-01

    We study theoretically the kinetics of mRNA translocation in the wild-type (WT) Escherichia coli ribosome, which is composed of a small 30S and large 50S subunit, and the ribosomes with mutations to some intersubunit bridges such as B1a, B4, B7a, and B8. The theoretical results reproduce well the available in vitro experimental data on the biphasic kinetics of the forward mRNA translocation catalyzed by elongation factor G (EF-G) hydrolyzing GTP, which can be best fit by the sum of two exponentials, and the monophasic kinetics of the spontaneous reverse mRNA translocation in the absence of the elongation factor, which can be best fit by a single-exponential function, in both the WT and mutant ribosomes. We show that both the mutation-induced increase in the maximal rate of the slow phase for the forward mRNA translocation and that in the rate of the spontaneous reverse mRNA translocation result from a reduction in the intrinsic energy barrier to resist the rotational movements between the two subunits, giving the same degree of increase in the two rates. The mutation-induced increase in the maximal rate of the fast phase for the forward mRNA translocation results mainly from the increase in the rate of the ribosomal unlocking, a conformational change in the ribosome that widens the mRNA channel for the mRNA translocation to take place, which could be partly due to the effect of the mutation on the intrasubunit 30S head rotation. Moreover, we study the translation rate of the WT and mutant ribosomes. It is shown that the translation rate versus the concentration of EF-G-GTP does not follow the Michaelis-Menten (MM) kinetics, which is in sharp contrast to the general property of other enzymes that the rate of the enzymatic reaction versus the concentration of a substrate follows the MM kinetics. The physical origin of this non-MM kinetics for the ribosome is revealed.

  2. Biphasic character of ribosomal translocation and non-Michaelis-Menten kinetics of translation

    NASA Astrophysics Data System (ADS)

    Xie, Ping

    2014-12-01

    We study theoretically the kinetics of mRNA translocation in the wild-type (WT) Escherichia coli ribosome, which is composed of a small 30 S and large 50 S subunit, and the ribosomes with mutations to some intersubunit bridges such as B1a, B4, B7a, and B8. The theoretical results reproduce well the available in vitro experimental data on the biphasic kinetics of the forward mRNA translocation catalyzed by elongation factor G (EF-G) hydrolyzing GTP, which can be best fit by the sum of two exponentials, and the monophasic kinetics of the spontaneous reverse mRNA translocation in the absence of the elongation factor, which can be best fit by a single-exponential function, in both the WT and mutant ribosomes. We show that both the mutation-induced increase in the maximal rate of the slow phase for the forward mRNA translocation and that in the rate of the spontaneous reverse mRNA translocation result from a reduction in the intrinsic energy barrier to resist the rotational movements between the two subunits, giving the same degree of increase in the two rates. The mutation-induced increase in the maximal rate of the fast phase for the forward mRNA translocation results mainly from the increase in the rate of the ribosomal unlocking, a conformational change in the ribosome that widens the mRNA channel for the mRNA translocation to take place, which could be partly due to the effect of the mutation on the intrasubunit 30S head rotation. Moreover, we study the translation rate of the WT and mutant ribosomes. It is shown that the translation rate versus the concentration of EF-G-GTP does not follow the Michaelis-Menten (MM) kinetics, which is in sharp contrast to the general property of other enzymes that the rate of the enzymatic reaction versus the concentration of a substrate follows the MM kinetics. The physical origin of this non-MM kinetics for the ribosome is revealed.

  3. Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

    PubMed Central

    Pavlov, Michael Y; Antoun, Ayman; Lovmar, Martin; Ehrenberg, Måns

    2008-01-01

    We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G. PMID:18497739

  4. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

    PubMed Central

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

  5. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  6. Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition

    PubMed Central

    Nüsslein, Klaus; Tiedje, James M.

    1998-01-01

    The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with several Clostridium assemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community. PMID:9546163

  7. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

    PubMed Central

    Connolly, Keith; Rife, Jason P.; Culver, Gloria

    2009-01-01

    Summary While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits (SSUs) and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of E. coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase deficient form of this protein. Our data reveals cold sensitivity and altered ribosomal profiles are associated with a ΔksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis prompting discussion of KsgA as a possible anti-microbial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family. PMID:18990185

  8. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA.

    PubMed

    Allen, P N; Noller, H F

    1989-08-05

    We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS

  9. Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.

    PubMed

    Márquez, Viter; Fröhlich, Thomas; Armache, Jean-Paul; Sohmen, Daniel; Dönhöfer, Alexandra; Mikolajka, Aleksandra; Berninghausen, Otto; Thomm, Michael; Beckmann, Roland; Arnold, Georg J; Wilson, Daniel N

    2011-02-04

    Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.

  10. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed Central

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-01-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome. PMID:321423

  11. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-03-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.

  12. Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

    SciTech Connect

    Zhou, Jie; Lancaster, Laura; Trakhanov, Sergei; Noller, Harry F.

    2012-03-26

    The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 {angstrom} crystal structure of the RF3 {center_dot} GDPNP {center_dot} ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7{sup o} rotation of the body and 14{sup o} rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

  13. Ribosomal Protein Methyltransferases in the Yeast Saccharomyces cerevisiae: Roles in Ribosome Biogenesis and Translation

    PubMed Central

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-01-01

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  14. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  15. Targeting ricin to the ribosome.

    PubMed

    May, Kerrie L; Yan, Qing; Tumer, Nilgun E

    2013-07-01

    The plant toxin ricin is highly toxic for mammalian cells and is of concern for bioterrorism. Ricin belongs to a family of functionally related toxins, collectively referred to as ribosome inactivating proteins (RIPs), which disable ribosomes and halt protein synthesis. Currently there are no specific antidotes against ricin or related RIPs. The catalytic subunit of ricin is an N-glycosidase that depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination activity inhibits translation and its biochemistry has been intensively studied. Yet, recent developments paint a more complex picture of toxicity, with ribosomal proteins and cellular signaling pathways contributing to the potency of ricin. In particular, several studies have now established the importance of the ribosomal stalk structure in facilitating the depurination activity and ribosome specificity of ricin and other RIPs. This review highlights recent developments defining toxin-ribosome interactions and examines the significance of these interactions for toxicity and therapeutic intervention.

  16. High-resolution structure of the Escherichia coli ribosome

    DOE PAGES

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

    2015-03-16

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

  17. Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

    PubMed

    Jakob, Steffen; Ohmayer, Uli; Neueder, Andreas; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Hochmuth, Eduard; Deutzmann, Rainer; Griesenbeck, Joachim; Tschochner, Herbert; Milkereit, Philipp

    2012-01-01

    Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

  18. Eukaryotic Ribosome Assembly and Nuclear Export.

    PubMed

    Nerurkar, Purnima; Altvater, Martin; Gerhardy, Stefan; Schütz, Sabina; Fischer, Ute; Weirich, Christine; Panse, Vikram Govind

    2015-01-01

    Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging. Assembly of the eukaryotic ribosome begins in the nucleolus and requires more than 350 conserved assembly factors, which transiently associate with the preribosome at specific maturation stages. After accomplishing their tasks, early-acting assembly factors are released, preparing preribosomes for nuclear export. Export competent preribosomal subunits are transported through nuclear pore complexes into the cytoplasm, where they undergo final maturation steps, which are closely connected to quality control, before engaging in translation. In this chapter, we focus on the final events that commit correctly assembled ribosomal subunits for translation.

  19. The translational fidelity function of IF3 during transition from the 30 S initiation complex to the 70 S initiation complex.

    PubMed

    Grigoriadou, Christina; Marzi, Stefano; Pan, Dongli; Gualerzi, Claudio O; Cooperman, Barry S

    2007-10-26

    IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.

  20. Nucleocytoplasmic transport of ribosomes in a eukaryotic system: Is there a facilitated transport process

    SciTech Connect

    Khanna-Gupta, A.; Ware, V.C. )

    1989-03-01

    The authors have examined the kinetics of the process by which ribosomes are exported from the nucleus to the cytoplasm using Xenopus laevis oocytes microinjected into the germinal vesicle with radiolabeled ribosomes or ribosomal subunits from X. laevis, Tetrahymena thermophila, or Escherichia coli. Microinjected eukaryotic mature ribosomes are redistributed into the oocyte cytoplasm by an apparent carrier-mediated transport process that exhibits saturation kinetics as increasing amounts of ribosomes are injected. T. thermophila ribosomes are competent to traverse the Xenopus nuclear envelope, suggesting that the basic mechanism underlying ribosome transport is evolutionarily conserved. Microinjected E. coli ribosomes are not transported in this system, indicating that prokaryotic ribosomes lack the signals required for transport. Surprisingly, coinjected small (40S) and large (60S) subunits from T. thermophila are transported significantly faster than individual subunits. These observations support a facilitated transport model for the translocation of ribosomal subunits as separate units across the nuclear envelope whereby the transport rate of 60S or 40S subunits is enhanced by the presence of the partner subunit. Although the basic features of the transport mechanism have been preserved through evolution, other aspects of the process may be mediated through species-specific interactions. They hypothesize that a species-specific nuclear 40S-60S subunit association may expedite the transport of individual subunits across the nuclear envelope.

  1. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

    PubMed Central

    Failmezger, Jurek; Nitschel, Robert; Sánchez-Kopper, Andrés; Kraml, Michael; Siemann-Herzberg, Martin

    2016-01-01

    Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems. PMID:27992588

  2. Ribosome dynamics and the evolutionary history of ribosomes

    NASA Astrophysics Data System (ADS)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  3. Import of ribosomal proteins into yeast mitochondria.

    PubMed

    Woellhaf, Michael W; Hansen, Katja G; Garth, Christoph; Herrmann, Johannes M

    2014-12-01

    Mitochondrial ribosomes of baker's yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences, suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and indeed showed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix, and thereby specifically focus on proteins of the mitochondrial ribosome.

  4. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

  5. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  6. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

  7. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    , the protein itself causes a feedback inhibition of the splicing of the transcript of its own gene. The synthesis of ribosomes involves a massive transfer of material across the nuclear envelope in both directions. Nuclear localization signals have been identified for at least three ribosomal proteins; they are similar but not identical to those identified for the simian virus 40 T antigen. There is no information about how ribosomal subunits are transported from the nucleus to the cytoplasm.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2666845

  8. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  9. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    NASA Astrophysics Data System (ADS)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  10. Error-prone and error-restrictive mutations affecting ribosomal protein S12.

    PubMed

    Agarwal, Deepali; Gregory, Steven T; O'Connor, Michael

    2011-07-01

    Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.

  11. Differential scanning calorimetry of whole Escherichia coli treated with the antimicrobial peptide MSI-78 indicate a multi-hit mechanism with ribosomes as a novel target.

    PubMed

    Brannan, Alexander M; Whelan, William A; Cole, Emma; Booth, Valerie

    2015-01-01

    Differential Scanning Calorimetry (DSC) of intact Escherichia coli (E. coli) was used to identify non-lipidic targets of the antimicrobial peptide (AMP) MSI-78. The DSC thermograms revealed that, in addition to its known lytic properties, MSI-78 also has a striking effect on ribosomes. MSI-78's effect on DSC scans of bacteria was similar to that of kanamycin, an antibiotic drug known to target the 30S small ribosomal subunit. An in vitro transcription/translation assay helped confirm MSI-78's targeting of ribosomes. The scrambled version of MSI-78 also affected the ribosome peak of the DSC scans, but required greater amounts of peptide to cause a similar effect to the unscrambled peptide. Furthermore, the effect of the scrambled peptide was not specific to the ribosomes; other regions of the DSC thermogram were also affected. These results suggest that MSI-78's effects on E. coli are at least somewhat dependent on its particular structural features, rather than a sole function of its overall charge and hydrophobicity. When considered along with earlier work detailing MSI-78's membrane lytic properties, it appears that MSI-78 operates via a multi-hit mechanism with multiple targets.

  12. Differential scanning calorimetry of whole Escherichia coli treated with the antimicrobial peptide MSI-78 indicate a multi-hit mechanism with ribosomes as a novel target

    PubMed Central

    Brannan, Alexander M.; Whelan, William A.; Cole, Emma

    2015-01-01

    Differential Scanning Calorimetry (DSC) of intact Escherichia coli (E. coli) was used to identify non-lipidic targets of the antimicrobial peptide (AMP) MSI-78. The DSC thermograms revealed that, in addition to its known lytic properties, MSI-78 also has a striking effect on ribosomes. MSI-78’s effect on DSC scans of bacteria was similar to that of kanamycin, an antibiotic drug known to target the 30S small ribosomal subunit. An in vitro transcription/translation assay helped confirm MSI-78’s targeting of ribosomes. The scrambled version of MSI-78 also affected the ribosome peak of the DSC scans, but required greater amounts of peptide to cause a similar effect to the unscrambled peptide. Furthermore, the effect of the scrambled peptide was not specific to the ribosomes; other regions of the DSC thermogram were also affected. These results suggest that MSI-78’s effects on E. coli are at least somewhat dependent on its particular structural features, rather than a sole function of its overall charge and hydrophobicity. When considered along with earlier work detailing MSI-78’s membrane lytic properties, it appears that MSI-78 operates via a multi-hit mechanism with multiple targets. PMID:26713257

  13. Studies on structural stability of thermophilic Sulfolobus acidocaldarius ribosomes.

    PubMed

    Yangala, Kalavathi; Suryanarayana, Tangirala

    2007-02-01

    Structural stability of thermophilic archaeon Sulfolobus acidocaldarius ribosomes, with respect their susceptibility to pancreatic RNase A and stability to temperature (deltaTm), on treatment with various stabilizing (polyamines) and destabilizing (sulfhydryl and intercalating) agents were studied and compared with mesophilic E. coli ribosomes, to understand the structural differences between thermophilic and mesophilic ribosomes. Thermophilic archaeal ribosomes and their subunits were 10-times less susceptible to pancreatic RNase A, compared to mesophilic ribosomes, showing the presence of strong and compact structural organization in them. Thermophilic ribosomes treated with destabilizing agents, such as sulfhydryl reagents [5,5'-Dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercurybenzoate) and intercalating agents (ethidium bromide, EtBr) showed higher stability to RNase A, compared to similarly treated mesophilic ribosomes, indicating the unavailability of thiol-reactive groups and the presence of strong solvent inaccessible inner core. Higher stability of thermophilic ribosomes compared to mesophilic ribosomes to unfolding agents like urea further supported the presence of strong inner core particle. Thermophilic ribosomes treated with intercalating agents, such as EtBr were less susceptible to RNase A, though they bound to more reagent, showing the rigidity or resilience of their macromolecular structure to alterations caused by destabilizing agents. Overall, these results indicated that factors such as presence of strong solvent inaccessible inner core and rigidity of ribosome macromolecular structure contributed stability of thermophilic ribosomes to RNase A and other destabilizing agents, when compared to mesophilic ribosomes.

  14. Bases in 16S rRNA Important for Subunit Association, tRNA binding, and Translocation

    PubMed Central

    Shi, Xinying; Chiu, Katie; Ghosh, Srikanta; Joseph, Simpson

    2009-01-01

    Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated in order to study their role in translation. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association and tRNA binding. In contrast, A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far away as 100 Å from the tRNA binding sites can be important for translation. PMID:19545171

  15. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    SciTech Connect

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada

    2010-04-26

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

  16. HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.

    PubMed

    Zhang, Yanqing; Mandava, Chandra Sekhar; Cao, Wei; Li, Xiaojing; Zhang, Dejiu; Li, Ningning; Zhang, Yixiao; Zhang, Xiaoxiao; Qin, Yan; Mi, Kaixia; Lei, Jianlin; Sanyal, Suparna; Gao, Ning

    2015-11-01

    Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

  17. How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography.

    PubMed Central

    Lim, V; Venclovas, C; Spirin, A; Brimacombe, R; Mitchell, P; Müller, F

    1992-01-01

    Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome. Images PMID:1614849

  18. Convergent evolution led ribosome inactivating proteins to interact with ribosomal stalk.

    PubMed

    Lapadula, Walter J; Sanchez-Puerta, M Virginia; Ayub, Maximiliano Juri

    2012-03-01

    Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by depurinating an adenine on the sarcin-ricin loop (SRL) of the large subunit ribosomal RNA. Several RIPs interact with the C-terminal end of ribosomal stalk P proteins, and this interaction is required for their full activity. In contrast, the activity of Pokeweed Antiviral Protein is not affected by blocking this stalk component. Here, we provide evidence from phylogenetic analyses and sequence alignments suggesting that the interaction with the C-terminal end of P proteins evolved independently in different RIPs by convergent evolution.

  19. SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli

    PubMed Central

    Singh, Navjot; Bubunenko, Mikhail; Smith, Carol; Abbott, David M.; Stringer, Anne M.; Shi, Ronald; Court, Donald L.

    2016-01-01

    ABSTRACT A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli. This complex is proposed to prevent Rho-dependent transcription termination by a process known as “antitermination.” The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex. PMID:26980831

  20. Structures of the Ribosome in Intermediate States of Ratcheting

    SciTech Connect

    Zhang, Wen; Dunkle, Jack A.; Cate, Jamie H.D.

    2009-10-21

    Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.

  1. Thermostability of mammalian brain ribosomes and the effects of nucleoside triphosphates on their heat-sensitivity.

    PubMed

    Grove, B K; Johnson, T C; Gilbert, B E

    1974-02-01

    Mammalian brain ribosomes were found to be heat-labile. On preincubation of the ribosomes at 37 degrees C, their ability to participate in polypeptide-synthesis reactions was substantially diminished. Despite the sensitivity of ribosomal protein synthesis to heat-inactivation, preincubation resulted in no significant alterations in ribosomal sedimentation profiles or changes in the integrity of the ribosomal RNA. The thermolability of brain ribosomes was shown to be associated with their inability to bind both template RNA and aminoacyl-tRNA. Similar experiments with brain ribosomal subunits demonstrated that the small (40S) subunit was more sensitive to heat-inactivation than the large (60S) subunit. The presence of ATP (1mm) protected ribosomes from thermal inactivation, although this protection was shown to be temporary. The protection appeared to be specific to nucleoside triphosphates, since GTP and UTP also stabilized ribosomes to thermal denaturation whereas nucleoside diphosphates (ADP) and nucleoside monophosphates (AMP and cyclic AMP) did not alter ribosomal sensitivity to heat. Although 1mm concentrations of nucleoside triphosphates protected ribosomes from heat-inactivation, the presence of higher concentrations resulted in complete inactivation of ribosomal activity.

  2. Isolation of Mitochondrial Ribosomes.

    PubMed

    Carroll, Adam J

    2017-01-01

    Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

  3. The Ribosome Filter Redux

    PubMed Central

    Mauro, Vincent P.; Edelman, Gerald M.

    2010-01-01

    The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it. PMID:17890902

  4. High-resolution structure of the Escherichia coli ribosome

    SciTech Connect

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; Altman, Roger B.; Blanchard, Scott C.; Cate, Jamie H. D.

    2015-03-16

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. In conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.

  5. Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli

    PubMed Central

    Di Pietro, Fabio; Brandi, Anna; Dzeladini, Nadire; Fabbretti, Attilio; Carzaniga, Thomas; Piersimoni, Lolita; Pon, Cynthia L; Giuliodori, Anna Maria

    2013-01-01

    Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock. PMID:23420694

  6. Networks of interactions in the secondary and tertiary structure of ribosomal RNA

    NASA Astrophysics Data System (ADS)

    Lee, Chang-Yong; Lee, Jung C.; Gutell, Robin R.

    2007-12-01

    We construct four different structural networks for both the secondary and tertiary structures of the 16S and 23S ribosomal RNAs (rRNAs) in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits, and investigate topological characteristics of the rRNA structures by determining relevant measures, such as the characteristic path length, the clustering coefficient, and the helix betweenness. This study reveals that the 23S rRNA network is more compact than the 16S rRNA networks, reflecting the more globular overall structure of the 23S rRNA relative to the 16S rRNA. In particular, the large number of tertiary interactions in the 23S rRNA tends to cluster, accounting for its small-world network properties. In addition, although the rRNA networks are not the scale-free network, their helix betweenness has a power-law distribution and is correlated with the phylogenetic conservation of helices. The higher the helix betweenness, the more conserved the helix. These results suggest a potential role of the rRNA network as a new quantitative approach in rRNA research.

  7. The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog, Rpl22l1

    PubMed Central

    O'Leary, Monique N.; Schreiber, Katherine H.; Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Hale, J. Scott; Academia, Emmeline C.; Shah, Shreya R.; Morton, John F.; Holstein, Carly A.; Martin, Dan B.; Kaeberlein, Matt; Ladiges, Warren C.; Fink, Pamela J.; MacKay, Vivian L.; Wiest, David L.; Kennedy, Brian K.

    2013-01-01

    Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog. PMID:23990801

  8. The SSU Processome in Ribosome Biogenesis – Progress and Prospects

    PubMed Central

    Phipps, Kathleen R.; Charette, J. Michael; Baserga, Susan J.

    2010-01-01

    The small subunit (SSU) processome is a 2.2 MDa ribonucleoprotein complex involved in the processing, assembly and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloguing the components is the first step towards understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into sub-complexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of sub-complex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole. PMID:21318072

  9. The Modular Adaptive Ribosome

    PubMed Central

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5’UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments. PMID:27812193

  10. The Modular Adaptive Ribosome.

    PubMed

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5'UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments.

  11. {sup 30}S Beam Development and X-ray Bursts

    SciTech Connect

    Kahl, D.; Kubono, S.; Binh, D. N.; Hashimoto, T.; Hayakawa, S.; Kurihara, Y.; Ohshiro, Y.; Yamaguchi, H.; Chen, A. A.; Chen, J.; Setoodeh nia, K.; Kaji, D.; Nishimura, S.; Kim, A.; Lee, N. H.; Wakabayashi, Y.

    2010-03-01

    Over the past three years, we have worked on developing a well-characterized {sup 30}S radioactive beam to be used in a future experiment aiming to directly measure to extrapolate the {sup 30}S(alpha,p) stellar reaction rate within the Gamow window of Type I X-ray bursts. The importance of the {sup 30}S(alpha,p) reaction to X-ray bursts is discussed. Given the astrophysical motivation, the successful results of and challenges involved in the production of a low-energy {sup 30}S beam are detailed. Finally, an overview of our future plans regarding this on-going project are presented.

  12. Measurement of internal movements of the Escherichia coli ribosome using Forster resonance energy transfer and microsecond, continuous-flow turbulent mixing in micro-fabricated devices

    NASA Astrophysics Data System (ADS)

    Majumdar, Zigurts Krishna

    We have studied internal movements of the Eschericia coli ribosome with Forster Resonance Energy Transfer (FRET) using multiple donor-acceptor pairs labeled at specific ribosomal protein residues. We have developed a novel methodology that allows a more quantitative interpretation of distance data from FRET measurements, accounting for specific effects when using fluorescent probes, such as: non-stoichiometric labeling when biochemical separation is not possible, quantification of static and dynamic quenching, changes in extinction coefficients, effects of the orientation factor and the presence of random and systematic errors. From the obtained distance data, 13 donor-acceptor positions (from 18 independent FRET pairs) are used to model internal movements within the 30S subunit upon 70S association. These measurements are also applied to monitoring inter-subunit movements in functional states of the ribosome that are associated with the translocation cycle of the ribosome. This work reveals internal movements of the ribosome observed for the first time in solution, and presents in vitro evidence for large concerted inter-subunit motions associated with ribosome translocation. The second half of this thesis is independent of the above. We present the design, construction and implementation of micro-fabricated, continuous-flow, turbulent mixing devices that can mix two or three fluids to complete homogeneity on the molecular scale in the microsecond range. The prototypical designs are compact, portable, simple to fabricate and consume smaller sample volumes than current technology. We characterize the turbulent mixing process in microfluidic channels with fluorescence intensity and lifetime imaging and show that both the dependence of mixing times and pressure drop on the flow velocity and Reynolds number agree well with theoretical expectations for turbulent pipe flow. The novelties in this work are: the new methods of fabrication which enable production of three

  13. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  14. Ribosome recycling: An essential process of protein synthesis.

    PubMed

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira

    2007-01-01

    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  15. Complete kinetic mechanism for recycling of the bacterial ribosome.

    PubMed

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

  16. Complete kinetic mechanism for recycling of the bacterial ribosome

    PubMed Central

    Borg, Anneli; Pavlov, Michael

    2016-01-01

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

  17. Eukaryotic ribosomes that lack a 5.8S RNA

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Woese, C. R.

    1986-01-01

    The 5.8S ribosomal RNA is believed to be a universal eukaryotic characteristic. It has no (size) counterpart among the prokaryotes, although its sequence is homologous with the first 150 or so nucleotides of the prokaryotic large subunit (23S) ribosomal RNA. An exception to this rule is reported here. The microsporidian Vairimorpha necatrix is a eukaryote that has no 5.8S rRNA. As in the prokaryotes, it has a single large subunit rRNA, whose 5-prime region corresponds to the 5.8S rRNA.

  18. Direct link between RACK1 function and localization at the ribosome in vivo.

    PubMed

    Coyle, Scott M; Gilbert, Wendy V; Doudna, Jennifer A

    2009-03-01

    The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-A crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the beta-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.

  19. Escherichia coli persister cells suppress translation by selectively disassembling and degrading their ribosomes.

    PubMed

    Cho, Junho; Rogers, Janet; Kearns, Mark; Leslie, Macall; Hartson, Steven D; Wilson, Kevin S

    2015-01-01

    Bacterial persisters are rare, phenotypically distinct cells that survive exposure to multiple antibiotics. Previous studies indicated that formation and maintenance of the persister phenotype are regulated by suppressing translation. To examine the mechanism of this translational suppression, we developed novel methodology to rapidly purify ribosome complexes from persister cells. We purified His-tagged ribosomes from Escherichia coli cells that over-expressed HipA protein, which induces persister formation, and were treated with ampicillin to remove antibiotic-sensitive cells. We profiled ribosome complexes and analyzed the ribosomal RNA and protein components from these persister cells. Our results show that (i) ribosomes in persisters exist largely as inactive ribosomal subunits, (ii) rRNAs and tRNAs are mostly degraded and (iii) a small fraction of the ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. Our findings explain the basis for translational suppression in persisters and suggest how persisters survive exposure to multiple antibiotics.

  20. Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

    SciTech Connect

    Nichols, C. E.; Johnson, C.; Lamb, H. K.; Lockyer, M.; Charles, I. G.; Hawkins, A. R.; Stammers, D. K.

    2007-11-01

    The X-ray crystal structure of the GTPase YjeQ from S. typhimurium is presented and compared with those of orthologues from T. maritima and B. subtilis. The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

  1. Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea

    PubMed Central

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-01-01

    We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis. PMID:11923331

  2. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea.

    PubMed

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-04-01

    We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

  3. The geometry of the ribosomal polypeptide exit tunnel.

    PubMed

    Voss, N R; Gerstein, M; Steitz, T A; Moore, P B

    2006-07-21

    The geometry of the polypeptide exit tunnel has been determined using the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The tunnel is a component of a much larger, interconnected system of channels accessible to solvent that permeates the subunit and is connected to the exterior at many points. Since water and other small molecules can diffuse into and out of the tunnel along many different trajectories, the large subunit cannot be part of the seal that keeps ions from passing through the ribosome-translocon complex. The structure referred to as the tunnel is the only passage in the solvent channel system that is both large enough to accommodate nascent peptides, and that traverses the particle. For objects of that size, it is effectively an unbranched tube connecting the peptidyl transferase center of the large subunit and the site where nascent peptides emerge. At no point is the tunnel big enough to accommodate folded polypeptides larger than alpha-helices.

  4. Vorticella Linnaeus, 1767 (Ciliophora, Oligohymenophora, Peritrichia) is a grade not a clade: redefinition of Vorticella and the families Vorticellidae and Astylozoidae using molecular characters derived from the gene coding for small subunit ribosomal RNA.

    PubMed

    Sun, Ping; Clamp, John; Xu, Dapeng; Kusuoka, Yasushi; Miao, Wei

    2012-01-01

    Recent phylogenetic analyses of the peritrich genus Vorticella have suggested that it might be paraphyletic, with one Vorticella species - Vorticella microstoma grouping with the swimming peritrichs Astylozoon and Opisthonecta in a distant clade. These results were based on very limited taxon sampling and thus could not be accepted as conclusive evidence for revising the generic classification. We tested paraphyly of the genus Vorticella by making a new analysis with a broad range of samples from three continents that yielded 52 new sequences of the gene coding for small subunit rRNA. Our results, together with the available sequences in Genbank, form a comprehensive set of data for the genus Vorticella. Analyses of these data showed that Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta form a well-supported, monophyletic clade, that is distinct from and basal to the family Vorticellidae containing other species of Vorticella. Paraphyly of the genus Vorticella and family Vorticellidae was strongly confirmed by these results. Furthermore, the two clades of Vorticella identified by the SSU rRNA gene are so genetically diverse whereas the genetic distances within the one containing Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta were so slight, which marked it as a separate family that must be defined by molecular characters in the absence of unifying morphological and morphogenetic characters. An emended characterization and status of the genus Vorticella, the families Vorticellidae and Astylozoidae are presented and discussed.

  5. A structural view on the mechanism of the ribosome-catalyzed peptide bond formation.

    PubMed

    Simonović, Miljan; Steitz, Thomas A

    2009-01-01

    The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.

  6. Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly.

    PubMed

    Hopple, J S; Vilgalys, R

    1999-10-01

    Phylogenetic relationships were investigated in the mushroom genus Coprinus based on sequence data from the nuclear encoded large-subunit rDNA gene. Forty-seven species of Coprinus and 19 additional species from the families Coprinaceae, Strophariaceae, Bolbitiaceae, Agaricaceae, Podaxaceae, and Montagneaceae were studied. A total of 1360 sites was sequenced across seven divergent domains and intervening sequences. A total of 302 phylogenetically informative characters was found. Ninety-eight percent of the average divergence between taxa was located within the divergent domains, with domains D2 and D8 being most divergent and domains D7 and D10 the least divergent. An empirical test of phylogenetic signal among divergent domains also showed that domains D2 and D3 had the lowest levels of homoplasy. Two equally most parsimonious trees were resolved using Wagner parsimony. A character-state weighted analysis produced 12 equally most parsimonious trees similar to those generated by Wagner parsimony. Phylogenetic analyses employing topological constraints suggest that none of the major taxonomic systems proposed for subgeneric classification is able to completely reflect phylogenetic relationships in Coprinus. A strict consensus integration of the two Wagner trees demonstrates the problematic nature of choosing outgroups within dark-spored mushrooms. The genus Coprinus is found to be polyphyletic and is separated into three distinct clades. Most Coprinus taxa belong to the first two clades, which together form a larger monophyletic group with Lacrymaria and Psathyrella in basal positions. A third clade contains members of Coprinus section Comati as well as the genus Leucocoprinus, Podaxis pistillaris, Montagnea arenaria, and Agaricus pocillator. This third clade is separated from the other species of Coprinus by members of the families Strophariaceae and Bolbitiaceae and the genus Panaeolus.

  7. Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

    PubMed Central

    Jiang, Yuan; Yang, Zhongqi; Wang, Xiaoyi; Hou, Yuxia

    2015-01-01

    The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus. PMID:25782000

  8. MPV17L2 is required for ribosome assembly in mitochondria

    PubMed Central

    Dalla Rosa, Ilaria; Durigon, Romina; Pearce, Sarah F.; Rorbach, Joanna; Hirst, Elizabeth M.A.; Vidoni, Sara; Reyes, Aurelio; Brea-Calvo, Gloria; Minczuk, Michal; Woellhaf, Michael W.; Herrmann, Johannes M.; Huynen, Martijn A.; Holt, Ian J.; Spinazzola, Antonella

    2014-01-01

    MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ0 cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid. PMID:24948607

  9. Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress.

    PubMed

    Kappeler, Kyle V; Zhang, Jack; Dinh, Thai Nho; Strom, Joshua G; Chen, Qin M

    2012-05-01

    Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

  10. Analysis of plant ribosomes with asymmetric flow field-flow fractionation.

    PubMed

    Pitkänen, Leena; Tuomainen, Päivi; Eskelin, Katri

    2014-02-01

    Ribosome profiling is a technique used to separate ribosomal subunits, 80S ribosomes (monosomes), and polyribosomes (polysomes) from other RNA-protein complexes. It is traditionally performed in sucrose gradients. In this study, we used asymmetric flow field-flow fractionation (AsFlFFF) to characterize ribosome profiles of Nicotiana benthamiana plants. With the optimized running conditions, we were able to separate free molecules from ribosomal subunits and intact ribosomes. We used various chemical and enzymatic treatments to validate the positions of subunits, monosomes, and polysomes in the AsFlFFF fractograms. We also characterized the protein and RNA content of AsFlFFF fractions by gel electrophoresis and western blotting. The reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ribosomes remained bound to messenger RNAs (mRNAs) during the analysis. Therefore, we conclude that AsFlFFF can be used for ribosome profiling to study the mRNAs that are being translated. It can also be used to study the protein composition of ribosomes that are active in translation at that particular moment.

  11. Eukaryotic Cells Producing Ribosomes Deficient in Rpl1 Are Hypersensitive to Defects in the Ubiquitin-Proteasome System

    PubMed Central

    McIntosh, Kerri B.; Bhattacharya, Arpita; Willis, Ian M.; Warner, Jonathan R.

    2011-01-01

    It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132. PMID:21858174

  12. Eukaryotic cells producing ribosomes deficient in Rpl1 are hypersensitive to defects in the ubiquitin-proteasome system.

    PubMed

    McIntosh, Kerri B; Bhattacharya, Arpita; Willis, Ian M; Warner, Jonathan R

    2011-01-01

    It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132.

  13. Ribosomal RNA sequence suggest microsporidia are extremely ancient eukaryotes

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Maddox, J. V.; Friedman, S.; Debrunner-Vossbrinck, B. A.; Woese, C. R.

    1987-01-01

    A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  14. 70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

    PubMed Central

    Yamamoto, Hiroshi; Wittek, Daniela; Gupta, Romi; Qin, Bo; Ueda, Takuya; Krause, Roland; Yamamoto, Kaori; Albrecht, Renate; Pech, Markus; Nierhaus, Knud H.

    2016-01-01

    According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism. PMID:26888283

  15. [Mg2+ ions affect the structure of the central domain of the 18S rRNA in the vicinity of the ribosomal protein S13 binding site].

    PubMed

    Ivanov, A V; Malygin, A A; Karpova, G G

    2013-01-01

    It is known that Mg2+ ions at high concentrations stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. Effect of Mg2+ on the formation of the 18S rRNA structure at the 40S subunit assembly remains poorly explored. In this paper, we show that the sequentional increase of the Mg2+ concentration from 0.5 mM to 20 mM leads to a significant decrease of the affinity of recombinant human ribosomal protein S13 (rpS13e) to a RNA transcript corresponding to the central domain fragment of the 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD (including the nucleotides of helices H20 and H22), whose availabilities to hydroxyl radicals were dependent on the Mg2+ concentration, were determined. It was found that increase of the concentrations of Mg2+ results in the enhanced accessibilities of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduces those of nucleotides A1023, A1024, and A1028-S1026 in the helix H20. Comparison of the results obtained with the crystallographic data on the structure of the central domain of 18S rRNA in the 40S ribosomal subunit led to conclusion that increase of Mg2+ concentrations results in the reorientation of helices H20 and H24 relatively helices H22 and H23 to form a structure, in which these helices are positioned the same way as in 40S subunits. Hence, saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding does, and leads to decreasing of this domain affinity to the protein.

  16. Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics.

    PubMed

    Gupta, Ankit; Shah, Priyanka; Haider, Afreen; Gupta, Kirti; Siddiqi, Mohammad Imran; Ralph, Stuart A; Habib, Saman

    2014-05-01

    Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.

  17. Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics

    PubMed Central

    Gupta, Ankit; Shah, Priyanka; Haider, Afreen; Gupta, Kirti; Siddiqi, Mohammad Imran; Ralph, Stuart A.; Habib, Saman

    2014-01-01

    Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins. PMID:24850912

  18. Identical ribosomal DNA sequence data from Pfiesteria piscicida (Dinophyceae) isolates with different toxicity phenotypes.

    PubMed

    Tengs, Torstein; Bowers, Holly A; Glasgow, Howard B; Burkholder, JoAnn M; Oldach, David W

    2003-09-01

    Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.

  19. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    PubMed Central

    Nilsson, Ola B.; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D.; O’Brien, Edward P.; Beckmann, Roland; von Heijne, Gunnar

    2015-01-01

    Summary At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. PMID:26321634

  20. Homoiterons and expansion in ribosomal RNAs

    PubMed Central

    Parker, Michael S.; Sallee, Floyd R.; Park, Edwards A.; Parker, Steven L.

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks. PMID:26636029

  1. Homoiterons and expansion in ribosomal RNAs.

    PubMed

    Parker, Michael S; Sallee, Floyd R; Park, Edwards A; Parker, Steven L

    2015-01-01

    Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

  2. Organization of proteins in mammalian mitochondrial ribosomes: accessibility to lactoperoxidase-catalyzed radioiodination

    SciTech Connect

    Denslow, N.D.; O'Brien, T.W.

    1984-08-10

    To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, double label iodination technique was used. Regions of r-proteins exposed in purified ribosomal subunits were labeled with /sup 131/I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with /sup 125/I using the chloramine-T mediated reaction. The ratio of /sup 131/I to /sup 125/I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the highly exposed group, and a large number of proteins in the intermediate exposure group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as assembly proteins, since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles.

  3. Choreography of molecular movements during ribosome progression along mRNA.

    PubMed

    Belardinelli, Riccardo; Sharma, Heena; Caliskan, Neva; Cunha, Carlos E; Peske, Frank; Wintermeyer, Wolfgang; Rodnina, Marina V

    2016-04-01

    During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

  4. Mutations Outside the Anisomycin-Binding Site Can Make Ribosomes Drug-Resistant

    SciTech Connect

    Blaha,G.; Gurel, G.; Schroeder, S.; Moore, P.; Steitz, T.

    2008-01-01

    Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.

  5. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  6. Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

    PubMed

    Hesketh, J E; Horne, Z; Campbell, G P

    1991-02-01

    Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

  7. Ribosome dynamics during decoding.

    PubMed

    Rodnina, Marina V; Fischer, Niels; Maracci, Cristina; Stark, Holger

    2017-03-19

    Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNA(Sec) and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.This article is part of the themed issue 'Perspectives on the ribosome'.

  8. Expression of ribosomal genes in pea cotyledons at the initial stages of germination

    SciTech Connect

    Gumilevskaya, N.A.; Chumikhina, L.V.; Akhmatova, A.T.; Kretovich, V.L.

    1986-01-20

    The time of appearance of newly synthesized rRNAs and ribosomal proteins (r-proteins) in the ribosomes of pea cotyledons (Pisum sativum L.) during germination was investigated. The ribosomal fraction was isolated and analyzed according to the method of germination of the embryo in the presence of labeled precursors or after pulse labeling of the embryos at different stages of germination. For the identification of newly synthesized rRNAs in the ribosomes we estimated the relative stability of labeled RNAs to the action of RNase, the sedimentation rate, the ability to be methylated in vivo in the presence of (/sup 14/C)CH/sub 3/-methionine, and the localization in the subunits of dissociated ribosomes. The presence of newly synthesized r-proteins in the ribosomes was judged on the basis of the electrophoretic similarity in SDS-disc electrophoresis of labeled polypeptides of purified ribosome preparations and of genuine r-proteins, as well as according to the localization of labeled proteins in the subunits of the dissociated ribosomes. It was shown that the expression of the ribosomal genes in highly specialized cells of pea cotyledons that have completed their growth occurs at very early stages of germination.

  9. The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering

    NASA Astrophysics Data System (ADS)

    Zaccai, Giuseppe; Natali, Francesca; Peters, Judith; Řihová, Martina; Zimmerman, Ella; Ollivier, J.; Combet, J.; Maurel, Marie-Christine; Bashan, Anat; Yonath, Ada

    2016-11-01

    Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the ‘lubricant’ for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome.

  10. The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering

    PubMed Central

    Zaccai, Giuseppe; Natali, Francesca; Peters, Judith; Řihová, Martina; Zimmerman, Ella; Ollivier, J.; Combet, J.; Maurel, Marie-Christine; Bashan, Anat; Yonath, Ada

    2016-01-01

    Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the ‘lubricant’ for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome. PMID:27849042

  11. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  12. Ribosomal Dynamics: Intrinsic Instability of a Molecular Machine

    NASA Astrophysics Data System (ADS)

    Gao, Haixiao; Le Barron, Jamie; Frank, Joachim

    Ribosomes are molecular machines that translate genetic message into nascent peptides, through a complex dynamics interplay with mRNAs, tRNAs, and various protein factors. A prominent example of ribosomal dynamics is the rotation of small ribosomal subunit with respect to a large subunit, characterized as the "ratchet motion," which is triggered by the binding of several translation factors. Here, we analyze two kinds of ribosomal ratchet motions, induced by the binding of EF-G and RF3, respectively, as previously observed by cryo-electron microscopy. Using the flexible fitting technique (real-space refinement) and an RNA secondary structure display tool (coloRNA), we obtained quasi-atomic models of the ribosome in these ratchet-motion-related functional states and mapped the observed differences onto the highly conserved RNA secondary structure. Comparisons between two sets of ratchet motions revealed that, while the overall patterns of the RNA displacement are very similar, several local regions stand out in their differential behavior, including the highly conserved GAC (GTPase-associated-center) region. We postulate that these regions are important in modulating general ratchet motion and bestowing it with the dynamic characteristics required for the specific function.

  13. Ribosome dynamics during decoding

    PubMed Central

    Maracci, Cristina; Stark, Holger

    2017-01-01

    Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNASec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation. This article is part of the themed issue ‘Perspectives on the ribosome’. PMID:28138068

  14. RiboVision suite for visualization and analysis of ribosomes.

    PubMed

    Bernier, Chad R; Petrov, Anton S; Waterbury, Chris C; Jett, James; Li, Fengbo; Freil, Larry E; Xiong, Xiao; Wang, Lan; Migliozzi, Blacki L R; Hershkovits, Eli; Xue, Yuzhen; Hsiao, Chiaolong; Bowman, Jessica C; Harvey, Stephen C; Grover, Martha A; Wartell, Zachary J; Williams, Loren Dean

    2014-01-01

    RiboVision is a visualization and analysis tool for the simultaneous display of multiple layers of diverse information on primary (1D), secondary (2D), and three-dimensional (3D) structures of ribosomes. The ribosome is a macromolecular complex containing ribosomal RNA and ribosomal proteins and is a key component of life responsible for the synthesis of proteins in all living organisms. RiboVision is intended for rapid retrieval, analysis, filtering, and display of a variety of ribosomal data. Preloaded information includes 1D, 2D, and 3D structures augmented by base-pairing, base-stacking, and other molecular interactions. RiboVision is preloaded with rRNA secondary structures, rRNA domains and helical structures, phylogeny, crystallographic thermal factors, etc. RiboVision contains structures of ribosomal proteins and a database of their molecular interactions with rRNA. RiboVision contains preloaded structures and data for two bacterial ribosomes (Thermus thermophilus and Escherichia coli), one archaeal ribosome (Haloarcula marismortui), and three eukaryotic ribosomes (Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens). RiboVision revealed several major discrepancies between the 2D and 3D structures of the rRNAs of the small and large subunits (SSU and LSU). Revised structures mapped with a variety of data are available in RiboVision as well as in a public gallery (). RiboVision is designed to allow users to distill complex data quickly and to easily generate publication-quality images of data mapped onto secondary structures. Users can readily import and analyze their own data in the context of other work. This package allows users to import and map data from CSV files directly onto 1D, 2D, and 3D levels of structure. RiboVision has features in rough analogy with web-based map services capable of seamlessly switching the type of data displayed and the resolution or magnification of the display. RiboVision is available at .

  15. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  16. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  17. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    SciTech Connect

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  18. Theoretical estimate of the effect of thermal agitation on ribosome motion generated by stochastic microswimming.

    PubMed

    González-García, José S

    2016-11-04

    The effect of thermal agitation on ribosome motion is evaluated through the Péclet number, assuming that the ribosome is self-propelled along the mRNA during protein synthesis by a swimming stroke consisting of a cycle of stochastically-generated ribosome configurations involving its two subunits. The ribosome velocity probability distribution function is obtained, giving an approximately normal distribution. Its mean and variance together with an estimate of the in vivo free diffusion coefficient of the ribosome and using only configuration changes of small size, give a Péclet number similar to motor proteins and microorganisms. These results suggest the feasibility of the stochastic microswimming hypothesis to explain ribosome motion.

  19. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    NASA Astrophysics Data System (ADS)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  20. A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei

    PubMed Central

    Fleming, Ian M. C.; Paris, Zdeněk; Gaston, Kirk W.; Balakrishnan, R.; Fredrick, Kurt; Rubio, Mary Anne T.; Alfonzo, Juan D.

    2016-01-01

    Most eukaryotic ribosomes contain 26/28S, 5S, and 5.8S large subunit ribosomal RNAs (LSU rRNAs) in addition to the 18S rRNA of the small subunit (SSU rRNA). However, in kinetoplastids, a group of organisms that include medically important members of the genus Trypanosoma and Leishmania, the 26/28S large subunit ribosomal RNA is uniquely composed of 6 rRNA fragments. In addition, recent studies have shown the presence of expansion segments in the large ribosomal subunit (60S) of Trypanosoma brucei. Given these differences in structure, processing and assembly, T. brucei ribosomes may require biogenesis factors not found in other organisms. Here, we show that one of two putative 3-methylcytidine methyltransferases, TbMTase37 (a homolog of human methyltransferase-like 6, METTL6), is important for ribosome stability in T. brucei. TbMTase37 localizes to the nucleolus and depletion of the protein results in accumulation of ribosomal particles lacking srRNA 4 and reduced levels of polysome associated ribosomes. We also find that TbMTase37 plays a role in cytokinesis, as loss of the protein leads to multi-flagellated and multi-nucleated cells. PMID:26888608

  1. Expanding the ribosomal universe.

    PubMed

    Dinman, Jonathan D; Kinzy, Terri Goss

    2009-12-09

    In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery.

  2. Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

    PubMed Central

    Ramanathan, Preethi; Matina, Tina; Wen, Jikai

    2013-01-01

    Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs. PMID:23638349

  3. Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress

    PubMed Central

    Russo, Annapina; Russo, Giulia

    2017-01-01

    The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways. PMID:28085118

  4. Structural signatures of antibiotic binding sites on the ribosome

    PubMed Central

    David-Eden, Hilda; Mankin, Alexander S.; Mandel-Gutfreund, Yael

    2010-01-01

    The ribosome represents a major target for antibacterial drugs. Being a complex molecular machine, it offers many potential sites for functional interference. The high-resolution structures of ribosome in complex with various antibiotics provide a unique data set for understanding the universal features of drug-binding pockets on the ribosome. In this work, we have analyzed the structural and evolutionary properties of 65 antibiotic binding sites (ABSs) in the ribosome. We compared these sites to similar-size computed pockets extracted from the small and large ribosomal subunits. Based on this analysis, we defined properties of the known drug-binding sites, which constitute the signature of a ‘druggable’ site. The most noticeable properties of the ABSs are prevalence of non-paired bases, a strong bias in favor of unusual syn conformation of the RNA bases and an unusual sugar pucker. We propose that despite the different geometric and chemical properties of diverse antibiotics, their binding sites tend to have common attributes that possibly reflect the potency of the pocket for binding small molecules. Finally, we utilized the ensemble of properties to derive a druggability index, which can be used in conjunction with site functionality information to identify new drug-binding sites on the ribosome. PMID:20494981

  5. Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system

    PubMed Central

    Sung, Min-Kyung; Reitsma, Justin M.; Sweredoski, Michael J.; Hess, Sonja; Deshaies, Raymond J.

    2016-01-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin–proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

  6. Isolation of ribosomes and polysomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

  7. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  8. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

    PubMed

    Wang, Minshi; Parshin, Andrey V; Shcherbik, Natalia; Pestov, Dimitri G

    2015-07-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

  9. Cytonuclear interactions and relaxed selection accelerate sequence evolution in organelle ribosomes.

    PubMed

    Sloan, Daniel B; Triant, Deborah A; Wu, Martin; Taylor, Douglas R

    2014-03-01

    Many mitochondrial and plastid protein complexes contain subunits that are encoded in different genomes. In animals, nuclear-encoded mitochondrial proteins often exhibit rapid sequence evolution, which has been hypothesized to result from selection for mutations that compensate for changes in interacting subunits encoded in mutation-prone animal mitochondrial DNA. To test this hypothesis, we analyzed nuclear genes encoding cytosolic and organelle ribosomal proteins in flowering plants. The model angiosperm genus Arabidopsis exhibits low organelle mutation rates, typical of most plants. Nevertheless, we found that (nuclear-encoded) subunits of organelle ribosomes in Arabidopsis have higher amino acid sequence polymorphism and divergence than their counterparts in cytosolic ribosomes, suggesting that organelle ribosomes experience relaxed functional constraint. However, the observed difference between organelle and cytosolic ribosomes was smaller than in animals and could be partially attributed to rapid evolution in N-terminal organelle-targeting peptides that are not involved in ribosome function. To test the role of organelle mutation more directly, we used transcriptomic data from an angiosperm genus (Silene) with highly variable rates of organelle genome evolution. We found that Silene species with unusually fast-evolving mitochondrial and plastid DNA exhibited increased amino acid sequence divergence in ribosomal proteins targeted to the organelles but not in those that function in cytosolic ribosomes. Overall, these findings support the hypothesis that rapid organelle genome evolution has selected for compensatory mutations in nuclear-encoded proteins. We conclude that coevolution between interacting subunits encoded in different genomic compartments within the eukaryotic cell is an important determinant of variation in rates of protein sequence evolution.

  10. Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

    PubMed Central

    Dang, H; Ellis, S R

    1990-01-01

    We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome. Images PMID:2263452

  11. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-06

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.

  12. Revisiting the Structures of Several Antibiotics Bound to the Bacterial Ribosome

    SciTech Connect

    D Bulkley; C Innis; G Blaha; T Steitz

    2011-12-31

    The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

  13. Revisiting the structures of several antibiotics bound to the bacterial ribosome

    SciTech Connect

    Bulkley, David; Innis, C. Axel; Blaha, Gregor; Steitz, Thomas A.

    2010-10-08

    The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

  14. Reconstitution of a Minimal Ribosome-Associated Ubiquitination Pathway with Purified Factors

    PubMed Central

    Shao, Sichen; Hegde, Ramanujan S.

    2014-01-01

    Summary Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway. PMID:25132172

  15. Assembling the archaeal ribosome: roles for translation-factor-related GTPases.

    PubMed

    Blombach, Fabian; Brouns, Stan J J; van der Oost, John

    2011-01-01

    The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a 'molecular switch' mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo-eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.

  16. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A.; Qian, Yu; Huang, Lan; Wang, Qi; Costello, Catherine E.; Aphasizhev, Ruslan

    2016-01-01

    Summary Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation. PMID:26713541

  17. The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation.

    PubMed

    Ning, Wei; Fei, Jingyi; Gonzalez, Ruben L

    2014-08-19

    One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines.

  18. Direct and high throughput (HT) interactions on the ribosomal surface by iRIA.

    PubMed

    Pesce, Elisa; Minici, Claudia; Baβler, Jochen; Hurt, Ed; Degano, Massimo; Calamita, Piera; Biffo, Stefano

    2015-10-21

    Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.

  19. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  20. Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

    SciTech Connect

    Harada, Y.; Li, H.; Li, Hua; Lennarz, W. J.

    2009-04-28

    Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.

  1. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  2. BALANCED PRODUCTION OF RIBOSOMAL PROTEINS

    PubMed Central

    Perry, Robert P.

    2017-01-01

    Eukaryotic ribosomes contain one molecule each of 79 different proteins. The genes encoding these proteins are usually at widely scattered loci and have distinctive promoters with certain common features. This minireview discusses the means by which cells manage to balance the production of ribosomal proteins so as to end up with equimolar quantities in the ribosome. Regulation at all levels of gene expression, from transcription to protein turnover, is considered. PMID:17689889

  3. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  4. Isolation of ribosomes by chromatography.

    PubMed

    Maguire, Bruce A

    2015-04-01

    Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

  5. Ribonuclease selection for ribosome profiling

    PubMed Central

    Gerashchenko, Maxim V.; Gladyshev, Vadim N.

    2017-01-01

    Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome–mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems. PMID:27638886

  6. Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

    PubMed Central

    Arenz, Stefan; Graf, Michael; Nguyen, Fabian; Huter, Paul; Polikanov, Yury S.; Blanchard, Scott C.; Wilson, Daniel N.

    2016-01-01

    The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis. PMID:27330110

  7. Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome.

    PubMed

    Arenz, Stefan; Juette, Manuel F; Graf, Michael; Nguyen, Fabian; Huter, Paul; Polikanov, Yury S; Blanchard, Scott C; Wilson, Daniel N

    2016-07-05

    The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

  8. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  9. Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

    PubMed

    Johnson, W

    1972-06-01

    The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

  10. Parallel Structural Evolution of Mitochondrial Ribosomes and OXPHOS Complexes.

    PubMed

    van der Sluis, Eli O; Bauerschmitt, Heike; Becker, Thomas; Mielke, Thorsten; Frauenfeld, Jens; Berninghausen, Otto; Neupert, Walter; Herrmann, Johannes M; Beckmann, Roland

    2015-04-09

    The five macromolecular complexes that jointly mediate oxidative phosphorylation (OXPHOS) in mitochondria consist of many more subunits than those of bacteria, yet, it remains unclear by which evolutionary mechanism(s) these novel subunits were recruited. Even less well understood is the structural evolution of mitochondrial ribosomes (mitoribosomes): while it was long thought that their exceptionally high protein content would physically compensate for their uniquely low amount of ribosomal RNA (rRNA), this hypothesis has been refuted by structural studies. Here, we present a cryo-electron microscopy structure of the 73S mitoribosome from Neurospora crassa, together with genomic and proteomic analyses of mitoribosome composition across the eukaryotic domain. Surprisingly, our findings reveal that both structurally and compositionally, mitoribosomes have evolved very similarly to mitochondrial OXPHOS complexes via two distinct phases: A constructive phase that mainly acted early in eukaryote evolution, resulting in the recruitment of altogether approximately 75 novel subunits, and a reductive phase that acted during metazoan evolution, resulting in gradual length-reduction of mitochondrially encoded rRNAs and OXPHOS proteins. Both phases can be well explained by the accumulation of (slightly) deleterious mutations and deletions, respectively, in mitochondrially encoded rRNAs and OXPHOS proteins. We argue that the main role of the newly recruited (nuclear encoded) ribosomal- and OXPHOS proteins is to provide structural compensation to the mutationally destabilized mitochondrially encoded components. While the newly recruited proteins probably provide a selective advantage owing to their compensatory nature, and while their presence may have opened evolutionary pathways toward novel mitochondrion-specific functions, we emphasize that the initial events that resulted in their recruitment was nonadaptive in nature. Our framework is supported by population genetic

  11. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    PubMed Central

    Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

    2012-01-01

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

  12. Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress.

    PubMed

    van den Elzen, Antonia M G; Schuller, Anthony; Green, Rachel; Séraphin, Bertrand

    2014-02-03

    Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosomes stabilized by the 'clamping' Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34-Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34-Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34-Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.

  13. Rapid cytoplasmic turnover of yeast ribosomes in response to rapamycin inhibition of TOR.

    PubMed

    Pestov, Dimitri G; Shcherbik, Natalia

    2012-06-01

    The target of rapamycin (TOR) pathway is the central regulator of cell growth in eukaryotes. Inhibition of TOR by rapamycin elicits changes in translation attributed mainly to altered translation initiation and repression of the synthesis of new ribosomes. Using quantitative analysis of rRNA, we found that the number of existing ribosomes present in a Saccharomyces cerevisiae culture during growth in rich medium rapidly decreases by 40 to 60% when the cells are treated with rapamycin. This process is not appreciably affected by a suppression of autophagy, previously implicated in degradation of ribosomes in eukaryotes upon starvation. Yeast cells deficient in the exosome function or lacking its cytoplasmic Ski cofactors show an abnormal pattern of rRNA degradation, particularly in the large ribosomal subunit, and accumulate rRNA fragments after rapamycin treatment and during diauxic shift. The exosome and Ski proteins are thus important for processing of rRNA decay intermediates, although they are probably not responsible for initiating rRNA decay. The role of cytoplasmic nucleases in rapamycin-induced rRNA degradation suggests mechanistic parallels of this process to nutrient-controlled ribosome turnover in prokaryotes. We propose that ribosome content is regulated dynamically in eukaryotes by TOR through both ribosome synthesis and the cytoplasmic turnover of mature ribosomes.

  14. Ribosomal Peptide Natural Products: Bridging the Ribosomal and Nonribosomal Worlds

    PubMed Central

    McIntosh, John A.; Donia, Mohamed S.; Schmidt, Eric W.

    2010-01-01

    Ribosomally synthesized bacterial natural products rival the nonribosomal peptides in their structural and functional diversity. The last decade has seen substantial progress in the identification and characterization of biosynthetic pathways leading to ribosomal peptide natural products with new and unusual structural motifs. In some of these cases, the motifs are similar to those found in nonribosomal peptides, and many are constructed by convergent or even paralogous enzymes. Here, we summarize the major structural and biosynthetic categories of ribosomally synthesized bacterial natural products and, where applicable, compare them to their homologs from nonribosomal biosynthesis. PMID:19642421

  15. Structural characterization of ribosome recruitment and translocation by type IV IRES.

    PubMed

    Murray, Jason; Savva, Christos G; Shin, Byung-Sik; Dever, Thomas E; Ramakrishnan, V; Fernández, Israel S

    2016-05-09

    Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

  16. Linezolid-Dependent Function and Structure Adaptation of Ribosomes in a Staphylococcus epidermidis Strain Exhibiting Linezolid Dependence

    PubMed Central

    Kokkori, Sofia; Apostolidi, Maria; Tsakris, Athanassios; Pournaras, Spyros

    2014-01-01

    Linezolid-dependent growth was recently reported in Staphylococcus epidermidis clinical strains carrying mutations associated with linezolid resistance. To investigate this unexpected behavior at the molecular level, we isolated active ribosomes from one of the linezolid-dependent strains and we compared them with ribosomes isolated from a wild-type strain. Both strains were grown in the absence and presence of linezolid. Detailed biochemical and structural analyses revealed essential differences in the function and structure of isolated ribosomes which were assembled in the presence of linezolid. The catalytic activity of peptidyltransferase was found to be significantly higher in the ribosomes derived from the linezolid-dependent strain. Interestingly, the same ribosomes exhibited an abnormal ribosomal subunit dissociation profile on a sucrose gradient in the absence of linezolid, but the profile was restored after treatment of the ribosomes with an excess of the antibiotic. Our study suggests that linezolid most likely modified the ribosomal assembly procedure, leading to a new functional ribosomal population active only in the presence of linezolid. Therefore, the higher growth rate of the partially linezolid-dependent strains could be attributed to the functional and structural adaptations of ribosomes to linezolid. PMID:24890589

  17. Linezolid-dependent function and structure adaptation of ribosomes in a Staphylococcus epidermidis strain exhibiting linezolid dependence.

    PubMed

    Kokkori, Sofia; Apostolidi, Maria; Tsakris, Athanassios; Pournaras, Spyros; Stathopoulos, Constantinos; Dinos, George

    2014-08-01

    Linezolid-dependent growth was recently reported in Staphylococcus epidermidis clinical strains carrying mutations associated with linezolid resistance. To investigate this unexpected behavior at the molecular level, we isolated active ribosomes from one of the linezolid-dependent strains and we compared them with ribosomes isolated from a wild-type strain. Both strains were grown in the absence and presence of linezolid. Detailed biochemical and structural analyses revealed essential differences in the function and structure of isolated ribosomes which were assembled in the presence of linezolid. The catalytic activity of peptidyltransferase was found to be significantly higher in the ribosomes derived from the linezolid-dependent strain. Interestingly, the same ribosomes exhibited an abnormal ribosomal subunit dissociation profile on a sucrose gradient in the absence of linezolid, but the profile was restored after treatment of the ribosomes with an excess of the antibiotic. Our study suggests that linezolid most likely modified the ribosomal assembly procedure, leading to a new functional ribosomal population active only in the presence of linezolid. Therefore, the higher growth rate of the partially linezolid-dependent strains could be attributed to the functional and structural adaptations of ribosomes to linezolid.

  18. The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

    NASA Astrophysics Data System (ADS)

    Bashan, Anat; Yonath, Ada

    2008-11-01

    Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to the large size, complex structure, inherent flexibility and high conformational variability of the ribosome. For the case of the small ribosomal subunit, which caused extreme difficulties, post-crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by "gluing" symmetrical related subunits. Here, we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment.

  19. Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites▿

    PubMed Central

    Basu, Abhijit; Das, Priyanka; Chaudhuri, Sujan; Bevilacqua, Elena; Andrews, Joel; Barik, Sailen; Hatzoglou, Maria; Komar, Anton A.; Mazumder, Barsanjit

    2011-01-01

    Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation. PMID:21930789

  20. Structure and Assembly Pathway of the Ribosome Quality Control Complex

    PubMed Central

    Shao, Sichen; Brown, Alan; Santhanam, Balaji; Hegde, Ramanujan S.

    2015-01-01

    Summary During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin’s N-terminal domain, while Listerin’s C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control. PMID:25578875

  1. The Structure of LepA, the Ribosomal Back Translocase

    SciTech Connect

    Evans,R.; Blaha, G.; Bailey, S.; Steitz, T.

    2008-01-01

    LepA is a highly conserved elongation factor that promotes the back translocation of tRNAs on the ribosome during the elongation cycle. We have determined the crystal structure of LepA from Escherichia coli at 2.8- Angstroms resolution. The high degree of sequence identity between LepA and EF-G is reflected in the structural similarity between the individual homologous domains of LepA and EF-G. However, the orientation of domains III and V in LepA differs from their orientations in EF-G. LepA also contains a C-terminal domain (CTD) not found in EF-G that has a previously unobserved protein fold. The high structural similarity between LepA and EF-G enabled us to derive a homology model for LepA bound to the ribosome using a 7.3- Angstroms cryo-EM structure of a complex between EF-G and the 70S ribosome. In this model, the very electrostatically positive CTD of LepA is placed in the direct vicinity of the A site of the large ribosomal subunit, suggesting a possible interaction between the CTD and the back translocated tRNA or 23S rRNA.

  2. The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome

    SciTech Connect

    Stanley, Robin E.; Blaha, Gregor; Grodzicki, Robert L.; Strickler, Michael D.; Steitz, Thomas A.

    2010-05-03

    Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.

  3. The Structure of the Anti-tuberculosis Antibiotics Viomycin and Capreomycin Bound to the 70S Ribosome

    SciTech Connect

    Stanley, R.; Blaha, G; Grodzicki, R; Strickler, M; Steitz, T

    2010-01-01

    Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.

  4. Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

    PubMed

    Mangiarotti, Giorgio

    2002-01-01

    Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.

  5. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

    PubMed Central

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  6. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.

  7. An Uncharged Amine in the Transition State of the Ribosomal Peptidyl Transfer Reaction

    PubMed Central

    Kingery, David A.; Pfund, Emmanuel; Voorhees, Rebecca M.; Okuda, Kensuke; Wohlgemuth, Ingo; Kitchen, David E.; Rodnina, Marina V.; Strobel, Scott A.

    2010-01-01

    SUMMARY The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the α-amino nucleophile (βnuc) using a series of puromycin derivatives. Both 50S subunit and 70S ribosome catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that at the transition state the nucleophile is neutral in the ribosome catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution. PMID:18482701

  8. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

    PubMed

    Ingolia, Nicholas T; Brar, Gloria A; Stern-Ginossar, Noam; Harris, Michael S; Talhouarne, Gaëlle J S; Jackson, Sarah E; Wills, Mark R; Weissman, Jonathan S

    2014-09-11

    Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

  9. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  10. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  11. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  12. Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation.

    PubMed

    Minakhina, Svetlana; Naryshkina, Tatyana; Changela, Neha; Tan, William; Steward, Ruth

    2016-01-01

    Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.

  13. Expanding the Entamoeba Universe: New Hosts Yield Novel Ribosomal Lineages.

    PubMed

    Jacob, Alison S; Busby, Eloise J; Levy, Abigail D; Komm, Natasha; Clark, C Graham

    2016-01-01

    Removing the requirement for cell culture has led to a substantial increase in the number of lineages of Entamoeba recognized as distinct. Surveying the range of potential host species for this parasite genus has barely been started and it is clear that additional sampling of the same host in different locations often identifies additional diversity. In this study, using small subunit ribosomal RNA gene sequencing, we identify four new lineages of Entamoeba, including the first report of Entamoeba from an elephant, and extend the host range of some previously described lineages. In addition, examination of microbiome data from a number of host animals suggests that substantial Entamoeba diversity remains to be uncovered.

  14. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

    NASA Astrophysics Data System (ADS)

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R.; Kim, Kelly H.; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T.; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-06-01

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes--UtpA and UtpB--interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

  15. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  16. The kissing-loop T-shaped structure translational enhancer of Pea enation mosaic virus can bind simultaneously to ribosomes and a 5' proximal hairpin.

    PubMed

    Gao, Feng; Gulay, Suna P; Kasprzak, Wojciech; Dinman, Jonathan D; Shapiro, Bruce A; Simon, Anne E

    2013-11-01

    The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.

  17. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  18. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon.

    PubMed

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S

    2015-06-15

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel.

  19. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  20. Challenges in describing ribosome dynamics

    NASA Astrophysics Data System (ADS)

    Nguyen, Kien; Whitford, Paul Charles

    2017-04-01

    For decades, protein folding and functional dynamics have been described in terms of diffusive motion across an underlying energy landscape. With continued advances in structural biology and high-performance computing, the field is positioned to extend these approaches to large biomolecular assemblies. Through the application of energy landscape techniques to the ribosome, one may work towards establishing a comprehensive description of the dynamics, which will bridge theoretical concepts and experimental observations. In this perspective, we discuss a few of the challenges that will need to be addressed as we extend the application of landscape principles to the ribosome.

  1. Ribosomal trafficking is reduced in Schwann cells following induction of myelination

    PubMed Central

    Love, James M.; Shah, Sameer B.

    2015-01-01

    Local synthesis of proteins within the Schwann cell periphery is extremely important for efficient process extension and myelination, when cells undergo dramatic changes in polarity and geometry. Still, it is unclear how ribosomal distributions are developed and maintained within Schwann cell projections to sustain local translation. In this multi-disciplinary study, we expressed a plasmid encoding a fluorescently labeled ribosomal subunit (L4-GFP) in cultured primary rat Schwann cells. This enabled the generation of high-resolution, quantitative data on ribosomal distributions and trafficking dynamics within Schwann cells during early stages of myelination, induced by ascorbic acid treatment. Ribosomes were distributed throughout Schwann cell projections, with ~2-3 bright clusters along each projection. Clusters emerged within 1 day of culture and were maintained throughout early stages of myelination. Three days after induction of myelination, net ribosomal movement remained anterograde (directed away from the Schwann cell body), but ribosomal velocity decreased to about half the levels of the untreated group. Statistical and modeling analysis provided additional insight into key factors underlying ribosomal trafficking. Multiple regression analysis indicated that net transport at early time points was dependent on anterograde velocity, but shifted to dependence on anterograde duration at later time points. A simple, data-driven rate kinetics model suggested that the observed decrease in net ribosomal movement was primarily dictated by an increased conversion of anterograde particles to stationary particles, rather than changes in other directional parameters. These results reveal the strength of a combined experimental and theoretical approach in examining protein localization and transport, and provide evidence of an early establishment of ribosomal populations within Schwann cell projections with a reduction in trafficking following initiation of myelination

  2. Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

    PubMed Central

    Folley, L. S.; Fox, T. D.

    1994-01-01

    A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products. PMID:8070651

  3. Defect in the formation of 70S ribosomes caused by lack of ribosomal protein L34 can be suppressed by magnesium.

    PubMed

    Akanuma, Genki; Kobayashi, Ako; Suzuki, Shota; Kawamura, Fujio; Shiwa, Yuh; Watanabe, Satoru; Yoshikawa, Hirofumi; Hanai, Ryo; Ishizuka, Morio

    2014-11-01

    To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ΔrpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ΔrpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg(2+), or overexpression of mgtE, which plays a major role in the import of Mg(2+), could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg(2+) content was lower in the ΔrpmH cells than in the wild type, and the Mg(2+) content in the ΔrpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg(2+). These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg(2+). In addition, the Mg(2+) content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ΔrplA (L1) and ΔrplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg(2+) content is influenced by the amount of 70S ribosomes.

  4. Evidence for a novel cytoplasmic processing event in ribosome maturation in the sea urchin Paracentrotus lividus.

    PubMed

    Bellavia, Daniele; Barbieri, Rainer

    2010-06-01

    In this paper we demonstrate the existence of a cytoplasmic processing step, never before described, involving both the pre-ribosomal subunits in the sea urchin Paracentrotus lividus. Northern-blot hybridization, primer extension, S1 mapping experiments and in situ hybridizations allowed us to demonstrate that cytoplasmic processed particles are successively re-imported into the nucleus where maturation of their RNAs is completed prior to being exported to the cytoplasm. Our findings lead to the proposal of a new model of ribosome maturation and shuttling.

  5. Chromatographic purification of highly active yeast ribosomes.

    PubMed

    Meskauskas, Arturas; Leshin, Jonathan A; Dinman, Jonathan D

    2011-10-24

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

  6. A new version of the RDP (Ribosomal Database Project)

    NASA Technical Reports Server (NTRS)

    Maidak, B. L.; Cole, J. R.; Parker, C. T. Jr; Garrity, G. M.; Larsen, N.; Li, B.; Lilburn, T. G.; McCaughey, M. J.; Olsen, G. J.; Overbeek, R.; Pramanik, S.; Schmidt, T. M.; Tiedje, J. M.; Woese, C. R.

    1999-01-01

    The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [ Nucleic Acids Res. (1997), 25, 109-111], is now hosted by the Center for Microbial Ecology at Michigan State University. RDP-II is a curated database that offers ribosomal RNA (rRNA) nucleotide sequence data in aligned and unaligned forms, analysis services, and associated computer programs. During the past two years, data alignments have been updated and now include >9700 small subunit rRNA sequences. The recent development of an ObjectStore database will provide more rapid updating of data, better data accuracy and increased user access. RDP-II includes phylogenetically ordered alignments of rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software programs for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (ftp.cme.msu. edu) and WWW (http://www.cme.msu.edu/RDP). The WWW server provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. Additional utilities also exist at RDP-II, including distance matrix, T-RFLP, and a Java-based viewer of the phylogenetic trees that can be used to create subtrees.

  7. {sup 30}S({alpha}, p) in X-Ray Bursts at CRIB

    SciTech Connect

    Kahl, D.; Kubono, S.; Binh, D. N.; Hashimoto, T.; Hayakawa, S.; Kurihara, Y.; Ohshiro, Y.; Yamaguchi, H.; Chen, A. A.; Chen, J.; Setoodeh nia, K.; Kaji, D.; Nishimura, S.; Kim, A.; Lee, N. H.; Wakabayashi, Y.

    2010-08-12

    Over the past three years, we have worked on developing a well-characterized {sup 30}S radioactive beam to be used in a future experiment aiming to directly measure the {sup 30}S({alpha}, p) stellar reaction rate within the Gamow window of Type I X-ray bursts.

  8. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  9. Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus.

    PubMed

    Stupina, Vera A; Yuan, Xuefeng; Meskauskas, Arturas; Dinman, Jonathan D; Simon, Anne E

    2011-05-01

    Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.

  10. All Ribosomes Are Created Equal. Really?

    PubMed

    Preiss, Thomas

    2016-02-01

    Ribosomes are generally thought of as molecular machines with a constitutive rather than regulatory role during protein synthesis. A study by Slavov et al.[1] now shows that ribosomes of distinct composition and functionality exist within eukaryotic cells, giving credence to the concept of 'specialized' ribosomes.

  11. Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes

    PubMed Central

    Zhang, Xing; Lai, Mason; Chang, Winston; Yu, Iris; Ding, Ke; Mrazek, Jan; Ng, Hwee L.; Yang, Otto O.; Maslov, Dmitri A.; Zhou, Z. Hong

    2016-01-01

    The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome. PMID:27752045

  12. Maize reas1 Mutant Stimulates Ribosome Use Efficiency and Triggers Distinct Transcriptional and Translational Responses1[OPEN

    PubMed Central

    Qi, Weiwei; Zhu, Jie; Wu, Qiao; Wang, Qun; Li, Xia; Yao, Dongsheng; Jin, Ying; Wang, Gang; Wang, Guifeng

    2016-01-01

    Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects; however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic initiation factor 2α and the increased elongation factor 1α level indicate an enhancement of general translational efficiency in dek* cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs. Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis. PMID:26645456

  13. Ribosomal accretion, apriorism and the phylogenetic method: a response to Petrov and Williams.

    PubMed

    Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

    2015-01-01

    Historical (ideographic) and non-historical (nomothetic) studies of ribosomal accretion appear to arrive at diametrically opposite conclusions. Phylogenetic analysis of thousands of RNA molecules and protein structures in hundreds of genomes supports the structural origin of the ribosome in RNA decoding and ribosomal mechanics. Predictions from extant features in a handful of rRNA structural models of the large ribosomal subunit support its origin in protein biosynthesis. In recent correspondence, one of us reported that correcting dismissals of conflicting data and avoiding unwarranted assumptions of the nomothetic method reconciled conclusions. In response, Petrov and Williams dismissed our arguments claiming we did not understand their algorithmic model of ribosomal apical growth. Instead, they controverted the historical approach. Here we show that their objections to the phylogenetic method are unjustified, that their algorithm subjectively guarantees back-in-time molecular deconstructions toward the protein biosynthetic core, and that processes of ribosomal growth are much more complex. We prompt abandoning apriorism, decreasing ad hoc hypotheses and integrating historical and non-historical scientific methods.

  14. Balanced Production of Ribosome Components Is Required for Proper G1/S Transition in Saccharomyces cerevisiae *

    PubMed Central

    Gómez-Herreros, Fernando; Rodríguez-Galán, Olga; Morillo-Huesca, Macarena; Maya, Douglas; Arista-Romero, María; de la Cruz, Jesús; Chávez, Sebastián; Muñoz-Centeno, Mari Cruz

    2013-01-01

    Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G1/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G1/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G1/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes. PMID:24043628

  15. Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria

    PubMed Central

    Wall, Erin A.; Caufield, J. Harry; Lyons, Charles E.; Manning, Keith A.; Dokland, Terje; Christie, Gail E.

    2015-01-01

    Summary Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway. PMID:25388641

  16. Nucleolin provides a link between RNA polymerase I transcription and pre-ribosome assembly.

    PubMed

    Roger, Benoit; Moisand, André; Amalric, François; Bouvet, Philippe

    2003-03-01

    Despite the identification of numerous factors involved in ribosomal RNA synthesis and maturation, the molecular mechanisms of ribosome biogenesis, and in particular the relationship between the different steps, are still largely unknown. We have investigated the consequences of an increased amount of a major nucleolar non-ribosomal protein, nucleolin, in Xenopus laevisstage VI oocytes on the production of ribosomal subunits. We show that a threefold increase in nucleolin leads to the complete absence of pre-rRNA maturation in addition to significant repression of RNA polymerase I transcription. Observation of "Christmas trees" by electron microscopy and analysis of the sedimentation properties of 40S pre-ribosomal particles suggest that an increased amount of nucleolin leads to incorrect packaging of the 40S particle. Interestingly, nucleolin affects the maturation of the 40S particle only when it is present at the time of transcription. These results indicate that nucleolin participates in the co-transcriptional packaging of the pre-rRNA, and that the quality of this packaging will determine whether the 40S precursor undergoes maturation or is degraded. The interaction of nucleolin with nascent pre-rRNA could help the co-transcriptional assembly on pre-rRNA of factors necessary for the subsequent maturation of the pre-ribosomal particle containing the 40S pre-rRNA.

  17. Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

    PubMed

    Wienen, B; Ehrlich, R; Stöffler-Meilicke, M; Stöffler, G; Smith, I; Weiss, D; Vince, R; Pestka, S

    1979-08-25

    Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

  18. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    SciTech Connect

    Feng, Yingang; Song, Xiaxia; Lin, Jinzhong; Xuan, Jinsong; Cui, Qiu; Wang, Jinfeng

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  19. Ribosomal accretion, apriorism and the phylogenetic method: a response to Petrov and Williams

    PubMed Central

    Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

    2015-01-01

    Historical (ideographic) and non-historical (nomothetic) studies of ribosomal accretion appear to arrive at diametrically opposite conclusions. Phylogenetic analysis of thousands of RNA molecules and protein structures in hundreds of genomes supports the structural origin of the ribosome in RNA decoding and ribosomal mechanics. Predictions from extant features in a handful of rRNA structural models of the large ribosomal subunit support its origin in protein biosynthesis. In recent correspondence, one of us reported that correcting dismissals of conflicting data and avoiding unwarranted assumptions of the nomothetic method reconciled conclusions. In response, Petrov and Williams dismissed our arguments claiming we did not understand their algorithmic model of ribosomal apical growth. Instead, they controverted the historical approach. Here we show that their objections to the phylogenetic method are unjustified, that their algorithm subjectively guarantees back-in-time molecular deconstructions toward the protein biosynthetic core, and that processes of ribosomal growth are much more complex. We prompt abandoning apriorism, decreasing ad hoc hypotheses and integrating historical and non-historical scientific methods. PMID:26082795

  20. Functional domains of the 50S subunit mature late in the assembly process

    PubMed Central

    Jomaa, Ahmad; Jain, Nikhil; Davis, Joseph H.; Williamson, James R.; Britton, Robert A.; Ortega, Joaquin

    2014-01-01

    Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit. PMID:24335279

  1. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

    PubMed Central

    Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  2. GTPases involved in bacterial ribosome maturation.

    PubMed

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  3. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  4. Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

    SciTech Connect

    Baram, David; Pyetan, Erez; Sittner, Assa; Auerbach-Nevo, Tamar; Bashan, Anat; Yonath, Ada

    2010-07-13

    Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-{angstrom} crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its 'signature motif' as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.

  5. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  6. Identification of previously unrecognized common elements in eukaryotic promoters. A ribosomal RNA gene initiator element for RNA polymerase I.

    PubMed

    Radebaugh, C A; Gong, X; Bartholomew, B; Paule, M R

    1997-02-07

    A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photo-cross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A133) to the rInr sequence. A185 also photo-cross-links when polymerase is stalled at +7.

  7. Characterization of hibernating ribosomes in mammalian cells.

    PubMed

    Krokowski, Dawid; Gaccioli, Francesca; Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A; Taylor, Derek; Hatzoglou, Maria

    2011-08-15

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.

  8. Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

    PubMed

    Makarov, G I; Golovin, A V; Sumbatyan, N V; Bogdanov, A A

    2015-08-01

    The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

  9. Ribosomal targets for antibiotic drug discovery

    DOEpatents

    Blanchard, Scott C.; Feldman, Michael Brian; Wang, Leyi; Doudna Cate, James H.; Pulk, Arto; Altman, Roger B.; Wasserman, Michael R

    2016-09-13

    The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

  10. Eukaryotic ribosome biogenesis at a glance.

    PubMed

    Thomson, Emma; Ferreira-Cerca, Sébastien; Hurt, Ed

    2013-11-01

    Ribosomes play a pivotal role in the molecular life of every cell. Moreover, synthesis of ribosomes is one of the most energetically demanding of all cellular processes. In eukaryotic cells, ribosome biogenesis requires the coordinated activity of all three RNA polymerases and the orchestrated work of many (>200) transiently associated ribosome assembly factors. The biogenesis of ribosomes is a tightly regulated activity and it is inextricably linked to other fundamental cellular processes, including growth and cell division. Furthermore, recent studies have demonstrated that defects in ribosome biogenesis are associated with several hereditary diseases. In this Cell Science at a Glance article and the accompanying poster, we summarise the current knowledge on eukaryotic ribosome biogenesis, with an emphasis on the yeast model system.

  11. The mitochondrial genome of the entomopathogenic fungus Beauveria bassiana: analysis of the ribosomal RNA region.

    PubMed

    Pfeifer, T A; Hegedus, D D; Khachatourians, G G

    1993-01-01

    The 28.5-kbp mitochondrial (mt) genome from the entomopathogenic fungus Beauveria bassiana was studied using restriction enzyme analysis, gene probe hybridization, and DNA sequence comparisons. A detailed restriction enzyme map allowed cloning of the entire genome into a number of segments. Hybridization of heterologous gene probes to the mtDNA resulted in the identification of the large ribosomal RNA (lrRNA) and small ribosomal RNA (srRNA) genes. Gene probes derived from several yeasts and fungi failed to identify any additional genes. However, partial DNA sequence analysis revealed the lrRNA and srRNA genes as well as four protein-encoding genes: the NADH dehydrogenase subunit 1 (NAD1), NADH dehydrogenase subunit 6 (NAD6), cytochrome oxidase subunit 3 (CO3), and ATPase subunit 6 (ATP6) genes. The ATPase subunit 9 (ATP9) gene was not identified by hybridization to mtDNA, but could be detected by hybridization to total cellular DNA. The portions of the genes sequenced were homologous to the equivalent genes from yeast and other filamentous fungi, most notably Aspergillus nidulans. No introns were identified in these regions. The organization of the sequenced region of the B. bassiana mt genome more closely resembled that of A. nidulans than that of Podospora anserina or Neurospora crassa.

  12. Physicochemical and Light Scattering Studies on Ribosome Particles

    PubMed Central

    Scafati, Anna Reale; Stornaiuolo, Maria Rosaria; Novaro, Patrizia

    1971-01-01

    The light scattering technique has been used to measure the molecular weight of Escherichia coli ribosomes. The 30S, 50S, and 70S components have been isolated and purified. The refractive index increment dn/dc was found to have the same value, (0.20 ±0.01) cm3/g, for the three species. The molecular weights are (1.0 ±0.1)·106, (1.7 ±0.1)·106, and (2.9 ±0.3)·106 daltons respectively. Some information about the dimensions in solution (radius of gyration) and the interaction constant (second virial coefficient) have been obtained, and their significance is discussed. PMID:4931397

  13. Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus

    PubMed Central

    Eyal, Zohar; Matzov, Donna; Krupkin, Miri; Wekselman, Itai; Paukner, Susanne; Zimmerman, Ella; Rozenberg, Haim; Bashan, Anat; Yonath, Ada

    2015-01-01

    The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus. PMID:26464510

  14. Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus.

    PubMed

    Eyal, Zohar; Matzov, Donna; Krupkin, Miri; Wekselman, Itai; Paukner, Susanne; Zimmerman, Ella; Rozenberg, Haim; Bashan, Anat; Yonath, Ada

    2015-10-27

    The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.

  15. Structural characterization of ribosome recruitment and translocation by type IV IRES

    PubMed Central

    Murray, Jason; Savva, Christos G; Shin, Byung-Sik; Dever, Thomas E; Ramakrishnan, V; Fernández, Israel S

    2016-01-01

    Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation. DOI: http://dx.doi.org/10.7554/eLife.13567.001 PMID:27159451

  16. Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II.

    PubMed

    Sierra, Raymond G; Gati, Cornelius; Laksmono, Hartawan; Dao, E Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S; Young, Iris D; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S; Koglin, Jason E; Boutet, Sébastien; Junco, Elia A; Hayes, Brandon; Bogan, Michael J; Hampton, Christina Y; Puglisi, Elisabetta V; Sauter, Nicholas K; Stan, Claudiu A; Zouni, Athina; Yano, Junko; Yachandra, Vittal K; Soltis, S Michael; Puglisi, Joseph D; DeMirci, Hasan

    2016-01-01

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

  17. Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

    SciTech Connect

    Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan; Dao, E. Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S.; Young, Iris D.; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S.; Koglin, Jason E.; Boutet, Sébastien; Junco, Elia A.; Hayes, Brandon; Bogan, Michael J.; Hampton, Christina Y.; Puglisi, Elisabetta V.; Sauter, Nicholas K.; Stan, Claudiu A.; Zouni, Athina; Yano, Junko; Yachandra, Vittal K.; Soltis, S. Michael; Puglisi, Joseph D.; DeMirci, Hasan

    2015-11-30

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

  18. The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

    PubMed Central

    Kurkcuoglu, Ozge; Doruker, Pemra; Sen, Taner Z.; Kloczkowski, Andrzej; Jernigan, Robert L.

    2009-01-01

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3’ end forming the Shine-Dalgarno complex at the initiation step; the 3’ end may act as a ‘hook’ to reel in the mRNA to facilitate its exit. PACS: 87.10.Pq; 87.15.bk; 87.15.kj; 87.16.dj; 87.16.dr PMID:19029596

  19. Nuclear structure of 30S and its implications for nucleosynthesis in classical novae

    NASA Astrophysics Data System (ADS)

    Setoodehnia, K.; Chen, A. A.; Kahl, D.; Komatsubara, T.; José, J.; Longland, R.; Abe, Y.; Binh, D. N.; Chen, J.; Cherubini, S.; Clark, J. A.; Deibel, C. M.; Fukuoka, S.; Hashimoto, T.; Hayakawa, T.; Hendriks, J.; Ishibashi, Y.; Ito, Y.; Kubono, S.; Lennard, W. N.; Moriguchi, T.; Nagae, D.; Nishikiori, R.; Niwa, T.; Ozawa, A.; Parker, P. D.; Seiler, D.; Shizuma, T.; Suzuki, H.; Wrede, C.; Yamaguchi, H.; Yuasa, T.

    2013-06-01

    Background: The uncertainty in the 29P(p,γ)30S reaction rate over 0.1 ≤ T ≤ 1.3 GK was previously determined to span approximately four orders of magnitude due to the uncertain location of two previously unobserved 3+ and 2+ resonances in the Ex=4.7-4.8 MeV region in 30S. Therefore, the abundances of silicon isotopes synthesized in novae, which are relevant for the identification of presolar grains of putative nova origin, were uncertain by a factor of 3.Purpose: (a) To investigate the level structure of 30S above the proton threshold [4394.9(7) keV] via charged-particle spectroscopy using the 32S(p,t)30S reaction and in-beam γ-ray spectroscopy using the 28Si(3He, nγ)30S reaction to calculate the 29P(p,γ)30S reaction rate. (b) To explore the impact of this rate on the abundances of silicon isotopes synthesized in novae.Methods: Differential cross sections of the 32S(p,t)30S reaction were measured at 34.5 MeV. Distorted-wave Born approximation calculations were performed to constrain the spin-parity assignments of the observed levels, including the two astrophysically important levels. An energy-level scheme was deduced from γ-γ coincidence measurements using the 28Si(3He, nγ)30S reaction. Spin-parity assignments based on measurements of γ-ray angular distributions and γ-γ directional correlation from oriented nuclei were made for most of the observed levels of 30S.Results: The resonance energies corresponding to the states with 4.5 MeV ≲ Ex ≲ 6 MeV, including the two astrophysically important states predicted previously, are measured with significantly better precision than before. The spin-parity assignments of both astrophysically important resonances are confirmed. The uncertainty in the rate of the 29P(p,γ)30S reaction is substantially reduced over the temperature range of interest. Finally, the influence of this rate on the abundance ratios of silicon isotopes synthesized in novae are obtained via 1D hydrodynamic nova simulations

  20. Distinct types of translation termination generate substrates for ribosome-associated quality control.

    PubMed

    Shcherbik, Natalia; Chernova, Tatiana A; Chernoff, Yury O; Pestov, Dimitri G

    2016-08-19

    Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45-Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.

  1. The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts.

    PubMed

    Wan, Kun; Kawara, Haruka; Yamamoto, Tomoyuki; Kume, Kazunori; Yabuki, Yukari; Goshima, Tetsuya; Kitamura, Kenji; Ueno, Masaru; Kanai, Muneyoshi; Hirata, Dai; Funato, Kouichi; Mizuta, Keiko

    2015-09-01

    The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.

  2. Distinct types of translation termination generate substrates for ribosome-associated quality control

    PubMed Central

    Shcherbik, Natalia; Chernova, Tatiana A.; Chernoff, Yury O.; Pestov, Dimitri G.

    2016-01-01

    Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45–Sup35 (eRF1–eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45–Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control. PMID:27325745

  3. Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

    PubMed Central

    Abeyrathne, Priyanka D; Koh, Cha San; Grant, Timothy; Grigorieff, Nikolaus; Korostelev, Andrei A

    2016-01-01

    Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation. DOI: http://dx.doi.org/10.7554/eLife.14874.001 PMID:27159452

  4. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin

    PubMed Central

    Harms, Jörg M; Schlünzen, Frank; Fucini, Paola; Bartels, Heike; Yonath, Ada

    2004-01-01

    Background The bacterial ribosome is a primary target of several classes of antibiotics. Investigation of the structure of the ribosomal subunits in complex with different antibiotics can reveal the mode of inhibition of ribosomal protein synthesis. Analysis of the interactions between antibiotics and the ribosome permits investigation of the specific effect of modifications leading to antimicrobial resistances. Streptogramins are unique among the ribosome-targeting antibiotics because they consist of two components, streptogramins A and B, which act synergistically. Each compound alone exhibits a weak bacteriostatic activity, whereas the combination can act bactericidal. The streptogramins A display a prolonged activity that even persists after removal of the drug. However, the mode of activity of the streptogramins has not yet been fully elucidated, despite a plethora of biochemical and structural data. Results The investigation of the crystal structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with the clinically relevant streptogramins quinupristin and dalfopristin reveals their unique inhibitory mechanism. Quinupristin, a streptogramin B compound, binds in the ribosomal exit tunnel in a similar manner and position as the macrolides, suggesting a similar inhibitory mechanism, namely blockage of the ribosomal tunnel. Dalfopristin, the corresponding streptogramin A compound, binds close to quinupristin directly within the peptidyl transferase centre affecting both A- and P-site occupation by tRNA molecules. Conclusions The crystal structure indicates that the synergistic effect derives from direct interaction between both compounds and shared contacts with a single nucleotide, A2062. Upon binding of the streptogramins, the peptidyl transferase centre undergoes a significant conformational transition, which leads to a stable, non-productive orientation of the universally conserved U2585. Mutations of this rRNA base are known to yield

  5. Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

    PubMed Central

    Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

    2016-01-01

    Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

  6. Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

    PubMed

    Nomura, Takaomi; Ito, Miho; Kanamori, Mai; Shigeno, Yuta; Uchiumi, Toshio; Arai, Ryoichi; Tsukada, Masuhiro; Hirabayashi, Kimio; Ohkawa, Kousaku

    2016-01-08

    Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.

  7. An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    PubMed Central

    Toribio, René; Díaz-López, Irene; Boskovic, Jasminka; Ventoso, Iván

    2016-01-01

    During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680–914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. PMID:26984530

  8. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  9. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  10. A two-step chemical mechanism for ribosome-catalysed peptide bond formation.

    PubMed

    Hiller, David A; Singh, Vipender; Zhong, Minghong; Strobel, Scott A

    2011-07-17

    The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups, lack of a dependence on pH, and the dominant contribution of entropy to catalysis, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2'-hydroxyl of the peptidyl-transfer RNA and a Brønsted coefficient near zero have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

  11. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs.

    PubMed

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-04-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

  12. Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Woolford, John L.; Baserga, Susan J.

    2013-01-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  13. Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

    PubMed Central

    Das, Priyanka; Basu, Abhijit; Biswas, Aditi; Poddar, Darshana; Andrews, Joel; Barik, Sailen; Komar, Anton A.

    2013-01-01

    In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis. PMID:23689135

  14. The Evolution of the Ribosome and the Genetic Code

    PubMed Central

    Hartman, Hyman; Smith, Temple F.

    2014-01-01

    The evolution of the genetic code is mapped out starting with the aminoacyl tRNA-synthetases and their interaction with the operational code in the tRNA acceptor arm. Combining this operational code with a metric based on the biosynthesis of amino acids from the Citric acid, we come to the conclusion that the earliest genetic code was a Guanine Cytosine (GC) code. This has implications for the likely earliest positively charged amino acids. The progression from this pure GC code to the extant one is traced out in the evolution of the Large Ribosomal Subunit, LSU, and its proteins; in particular those associated with the Peptidyl Transfer Center (PTC) and the nascent peptide exit tunnel. This progression has implications for the earliest encoded peptides and their evolutionary progression into full complex proteins. PMID:25370196

  15. The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction.

    PubMed Central

    Wu, H; Jiang, L; Zimmermann, R A

    1994-01-01

    Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions. PMID:7515489

  16. Kinetic analysis of pre-ribosome structure in vivo

    PubMed Central

    Swiatkowska, Agata; Wlotzka, Wiebke; Tuck, Alex; Barrass, J. David; Beggs, Jean D.; Tollervey, David

    2012-01-01

    Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3′ end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA–protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA–18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3′ guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA–protein complex are potentially amenable to this approach. PMID:23093724

  17. Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

    PubMed

    Archer, Stuart K; Shirokikh, Nikolay E; Beilharz, Traude H; Preiss, Thomas

    2016-07-28

    Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences

  18. The other lives of ribosomal proteins

    PubMed Central

    2010-01-01

    Despite the fact that ribosomal proteins are the constituents of an organelle that is present in every cell, they show a surprising level of regulation, and several of them have also been shown to have other extra-ribosomal functions, such in replication, transcription, splicing or even ageing. This review provides a comprehensive summary of these important aspects. PMID:20650820

  19. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  20. Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2).

    PubMed

    Lackmann, Fredrik; Belikov, Sergey; Wieslander, Lars

    2017-01-01

    Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S rRNA of the small ribosomal subunit. Mrd1p is evolutionary conserved in all eukaryotes and in yeast it contains five RNA Binding Domains (RBDs) separated by linker regions. One of these linkers, Linker 2 between RBD2 and RBD3, is conserved in length, predicted to be structured and contains conserved clusters of amino acid residues. In this report, we have analysed Linker 2 mutations and demonstrate that it is essential for Mrd1p function during pre-ribosomal processing. Extensive changes of amino acid residues as well as specific changes of conserved clusters of amino acid residues were found to be incompatible with synthesis of pre-40S ribosomes and cell growth. In addition, gross changes in primary sequence of Linker 2 resulted in Mrd1p instability, leading to degradation of the N-terminal part of the protein. Our data indicates that Linker 2 is functionally coupled to RBD2 and argues for that these domains constitute a functional module in Mrd1p. We conclude that Linker 2 has an essential role for Mrd1p beyond just providing a defined length between RBD2 and RBD3.

  1. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  2. Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

    PubMed

    Tereshchenkov, A G; Shishkina, A V; Tashlitsky, V N; Korshunova, G A; Bogdanov, A A; Sumbatyan, N V

    2016-04-01

    Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

  3. Relationship between anaerobic capacity estimated using a single effort and 30-s tethered running outcomes

    PubMed Central

    Miyagi, Willian Eiji; Sousa, Filipe Antônio de Barros; Gobatto, Claudio Alexandre

    2017-01-01

    The purpose of the current study was to investigate the relationship between alternative anaerobic capacity method (MAODALT) and a 30-s all-out tethered running test. Fourteen male recreational endurance runners underwent a graded exercise test, a supramaximal exhaustive effort and a 30-s all-out test on different days, interspaced by 48h. After verification of data normality (Shapiro-Wilk test), the Pearson’s correlation test was used to verify the association between the anaerobic estimates from the MAODALT and the 30-s all-out tethered running outputs. Absolute MAODALT was correlated with mean power (r = 0.58; P = 0.03), total work (r = 0.57; P = 0.03), and mean force (r = 0.79; P = 0.001). In addition, energy from the glycolytic pathway (E[La-]) was correlated with mean power (r = 0.58; P = 0.03). Significant correlations were also found at each 5s interval between absolute MAODALT and force values (r between 0.75 and 0.84), and between force values and E[La-] (r between 0.73 to 0.80). In conclusion, the associations between absolute MAODALT and the mechanical outputs from the 30-s all-out tethered running test evidenced the importance of the anaerobic capacity for maintaining force during the course of time in short efforts. PMID:28182775

  4. Quantitative studies of mRNA recruitment to the eukaryotic ribosome

    PubMed Central

    Fraser, Christopher S.

    2015-01-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5′ UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5′ UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. PMID:25742741

  5. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  6. Conjugates of amino acids and peptides with 5-o-mycaminosyltylonolide and their interaction with the ribosomal exit tunnel.

    PubMed

    Shishkina, Anna; Makarov, Gennady; Tereshchenkov, Andrey; Korshunova, Galina; Sumbatyan, Nataliya; Golovin, Andrey; Svetlov, Maxim; Bogdanov, Alexey

    2013-11-20

    During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

  7. Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates

    PubMed Central

    Lorenzi, Isotta; Oeljeklaus, Silke; Ronsör, Christin; Bareth, Bettina; Warscheid, Bettina; Dennerlein, Sven

    2016-01-01

    The three conserved core subunits of the cytochrome c oxidase are encoded by mitochondria in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing the N and C termini to the intermembrane space. For this, the N terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae. Little is known about the translocation of the C terminus, but Cox18 has been identified to be a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner. The Mba1-Cox20 complex accumulates when export of the C terminus of Cox2 is blocked by the loss of the Cox18 protein. While Cox20 engages with Cox18, Mba1 is no longer present at this stage. Our analyses indicate that Cox20 associates with nascent Cox2 and Mba1 to promote Cox2 maturation cotranslationally. We suggest that Mba1 stabilizes the Cox20-ribosome complex and supports the handover of Cox2 to the Cox18 tail export machinery. PMID:27550809

  8. Stepwise binding of tylosin and erythromycin to Escherichia coli ribosomes, characterized by kinetic and footprinting analysis.

    PubMed

    Petropoulos, Alexandros D; Kouvela, Ekaterini C; Dinos, George P; Kalpaxis, Dimitrios L

    2008-02-22

    Erythromycin and tylosin are 14- and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg2+ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg2+ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.

  9. Simulating movement of tRNA through the ribosome during hybrid-state formation

    NASA Astrophysics Data System (ADS)

    Whitford, Paul C.; Sanbonmatsu, Karissa Y.

    2013-09-01

    Biomolecular simulations provide a means for exploring the relationship between flexibility, energetics, structure, and function. With the availability of atomic models from X-ray crystallography and cryoelectron microscopy (cryo-EM), and rapid increases in computing capacity, it is now possible to apply molecular dynamics (MD) simulations to large biomolecular machines, and systematically partition the factors that contribute to function. A large biomolecular complex for which atomic models are available is the ribosome. In the cell, the ribosome reads messenger RNA (mRNA) in order to synthesize proteins. During this essential process, the ribosome undergoes a wide range of conformational rearrangements. One of the most poorly understood transitions is translocation: the process by which transfer RNA (tRNA) molecules move between binding sites inside of the ribosome. The first step of translocation is the adoption of a "hybrid" configuration by the tRNAs, which is accompanied by large-scale rotations in the ribosomal subunits. To illuminate the relationship between these rearrangements, we apply MD simulations using a multi-basin structure-based (SMOG) model, together with targeted molecular dynamics protocols. From 120 simulated transitions, we demonstrate the viability of a particular route during P/E hybrid-state formation, where there is asynchronous movement along rotation and tRNA coordinates. These simulations not only suggest an ordering of events, but they highlight atomic interactions that may influence the kinetics of hybrid-state formation. From these simulations, we also identify steric features (H74 and surrounding residues) encountered during the hybrid transition, and observe that flexibility of the single-stranded 3'-CCA tail is essential for it to reach the endpoint. Together, these simulations provide a set of structural and energetic signatures that suggest strategies for modulating the physical-chemical properties of protein synthesis by the

  10. Quantitative determination of ribosome nascent chain stability

    PubMed Central

    Samelson, Avi J.; Jensen, Madeleine K.; Soto, Randy A.; Cate, Jamie H. D.; Marqusee, Susan

    2016-01-01

    Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein’s energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome. PMID:27821780

  11. A new system for naming ribosomal proteins.

    PubMed

    Ban, Nenad; Beckmann, Roland; Cate, Jamie H D; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis L J; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian M T; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2014-02-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

  12. Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

    PubMed

    Hubbs, A E; Roy, H

    1993-06-25

    Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

  13. Centers of motion associated with EF-Tu binding to the ribosome.

    PubMed

    Paci, Maxim; Fox, George E

    2016-05-03

    Structural centers of motion (pivot points) in the ribosome have recently been identified by measurement of conformational changes in rRNA resulting from EF-G GTP hydrolysis. This series of measurements is extended here to the ribosome's interactions with the cofactor EF-Tu. Four recent EF-Tu bound ribosome structures were compared to unbound structures. A total of 16 pivots were identified, of which 4 are unique to the EF-Tu interaction. Pivots in the GTPase associated center and the sarcin-ricin loop omitted previously, are found to be mobile in response to both EF-Tu and EF-G binding. Pivots in the intersubunit bridge rRNAs are found to be cofactor specific. Head swiveling motions in the small subunit are observed in the EF-Tu bound structures that were trapped post GTP hydrolysis. As in the case of pivots associated with EF-G, the additional pivots described here are associated with weak points in the rRNA structures such as non-canonical pairs and bulge loops. The combined set of pivots should be regarded as a minimal set. Only several states available to the ribosome have been presented in this work. Future, precise crystal structures in conjunction with experimental data will likely show additional functional pivoting elements in the rRNA.

  14. Protein Folding Activity of the Ribosome (PFAR) –– A Target for Antiprion Compounds

    PubMed Central

    Banerjee, Debapriya; Sanyal, Suparna

    2014-01-01

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools. PMID:25341659

  15. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    PubMed Central

    Miller, Jennifer L.; Cimen, Huseyin; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially-encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCδ, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications. PMID:19702336

  16. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  17. Altered chlorplast ribosomal proteins associated with erythromycin-resistant mutants in two genetic systems of Chlamydomonas reinhardi.

    PubMed

    Mets, L; Bogorad, L

    1972-12-01

    The phenotype of several erythromycin-resistant mutants of Chlamydomonas reinhardi was further characterized in terms of the electrophoretic properties of their chloroplast ribosomal proteins. In mutant ery-M2d a single protein of the large (52 S) subunit has altered properties, which probably result from a change in its primary sequence. This mutation is inherited in a Meudelian manner. In mutant ery-U1a, which is inherited in a uniparental manner, a different single protein of the 52 S subunit is altered. This change might result from a change in either the primary sequence of the protein or in some form of secondary modification. These results indicate that these two distinct genetic systems must cooperate in the production of chloroplast ribosomes.

  18. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    PubMed

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

  19. The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

    PubMed

    Chirgadze, Yuri N; Clarke, Teresa E; Romanov, Vladimir; Kisselman, Gera; Wu-Brown, Jean; Soloveychik, Maria; Chan, Tiffany S Y; Gordon, Roni D; Battaile, Kevin P; Pai, Emil F; Chirgadze, Nickolay Y

    2015-02-01

    The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

  20. Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

    PubMed

    Pesaresi, P; Varotto, C; Meurer, J; Jahns, P; Salamini, F; Leister, D

    2001-08-01

    The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

  1. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  2. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon

    PubMed Central

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S.

    2015-01-01

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome–Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S–RQC and 80S–Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome–translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. PMID:25877867

  3. 30S(α , p) Thermonuclear Reaction Rate from Experimental Level Structure of 34Ar

    NASA Astrophysics Data System (ADS)

    Kahl, D.; Chen, A. A.; Kubono, S.; Yamaguchi, H.; Binh, D. N.; Chen, J.; Cherubini, S.; Duy, N. N.; Hashimoto, T.; Hayakawa, S.; Iwasa, N.; Jung, H. S.; Kato, S.; Kwon, Y. K.; Nishimura, S.; Ota, S.; Setoodehnia, K.; Teranishi, T.; Tokieda, H.; Yamada, T.; Yun, C. C.; Zhang, L. Y.

    Type I X-ray bursts are the most frequent thermonuclear explosions in the galaxy. Owing to their recurrence from known astronomical objects, burst morphology is extensively documented, and they are modeled very successfully as neutron-deficient, thermonuclear runaway on the surface of accreting neutron stars. While reaction networks include hundreds of isotopes and thousands of nuclear processes, only a small subset appear to play a pivotal role. One such reaction is the 30S(α , p) reaction, which is believed to be a crucial link in the explosive helium burning which is responsible for the large energy flux. However, very little experimental information is available concerning the cross section itself, nor the 34Ar compound nucleus at the relevant energies. We performed the first study of the entrance channel via 30S alpha resonant elastic scattering using a state-of-the-art, low-energy, 30S radioactive ion beam. The measurement was performed in inverse kinematics using a newly-developed active target. An R-matrix analysis of the excitation function reveals previously unknown resonances, including their quantum properties of spin, parity, width, and energy.

  4. Quantitative studies of ribosome conformational dynamics.

    PubMed

    Fraser, Christopher S; Doudna, Jennifer A

    2007-05-01

    The ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

  5. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-08-22

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome o