scholarly journals The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution

1982 ◽  
Vol 205 (3) ◽  
pp. 495-501 ◽  
Author(s):  
K Donceel ◽  
P Nieuwenhuysen ◽  
J Clauwaert
Keyword(s):  
Ribosomal Rna ◽  
Diffusion Coefficients ◽  
Brine Shrimp ◽  
Ribosomal Proteins ◽  
Hydrodynamic Radius ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Rrna Species ◽  
Reported Data ◽  
And Diffusion

The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.

Control points in eucaryotic ribosome biogenesis

1991 ◽  
Vol 69 (1) ◽  
pp. 5-22 ◽  
Author(s):  
D. E. Larson ◽  
P. Zahradka ◽  
B. H. Sells
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Polymerase Iii ◽  
Ribosomal Subunits ◽  
Rrna Species ◽  
Polymerase I ◽  
Ribosomal Precursor ◽  
Precursor Molecules

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

2021 ◽  
Author(s):  
Haina Huang ◽  
Melissa Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

scholarly journals A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1973 ◽  
Vol 133 (4) ◽  
pp. 739-747 ◽  
Author(s):  
A. Robinson ◽  
J. Sykes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Protein Loss ◽  
E Coli ◽  
Rrna Species ◽  
Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

scholarly journals Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis

2010 ◽  
Vol 30 (23) ◽  
pp. 5562-5571 ◽  
Author(s):  
Arpita Bhattacharya ◽  
Kerri B. McIntosh ◽  
Ian M. Willis ◽  
Jonathan R. Warner
Keyword(s):  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Slow Growth ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Growth Defects ◽  
Structural Problems ◽  
Genome Wide ◽  
40S Ribosomal Subunit ◽  
Severe Shortage

ABSTRACT A set of genome-wide screens for proteins whose absence exacerbates growth defects due to pseudo-haploinsufficiency of ribosomal proteins in Saccharomyces cerevisiae identified Dom34 as being particularly important for cell growth when there is a deficit of 40S ribosomal subunits. In contrast, strains with a deficit of 60S ribosomal proteins were largely insensitive to the loss of Dom34. The slow growth of cells lacking Dom34 and haploinsufficient for a protein of the 40S subunit is caused by a severe shortage of 40S subunits available for translation initiation due to a combination of three effects: (i) the natural deficiency of 40S subunits due to defective synthesis, (ii) the sequestration of 40S subunits due to the large accumulation of free 60S subunits, and (iii) the accumulation of ribosomes “stuck” in a distinct 80S form, insensitive to the Mg2+ concentration, and at least temporarily unavailable for further translation. Our data suggest that these stuck ribosomes have neither mRNA nor tRNA. We postulate, based on our results and on previously published work, that the stuck ribosomes arise because of the lack of Dom34, which normally resolves a ribosome stalled due to insufficient tRNAs, to structural problems with its mRNA, or to a defect in the ribosome itself.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽  
2022 ◽  
pp. rna.078994.121
Author(s):  
Haina Huang ◽  
Melissa D Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

scholarly journals Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariam Jaafar ◽  
Julia Contreras ◽  
Carine Dominique ◽  
Sara Martín-Villanueva ◽  
Régine Capeyrou ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rna Helicase ◽  
Genetic Interactions ◽  
Large Ribosomal Subunit ◽  
Base Pairing ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center

AbstractSynthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Electron Microscopy of EDTA-Treated Ribosomal Subunits from Escherichia coli

1971 ◽  
Vol 29 ◽  
pp. 432-433
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Gradient Analysis ◽  
Ribosomal Subunit ◽  
Nuclease Activity ◽  
Final Concentration ◽  
Ribosomal Subunits ◽  
Magnesium Removal ◽  
Low Ionic Strength ◽  
Endogenous Nuclease

Several studies have indicated that treatment of ribosomes and ribosomal subunits with EDTA at low ionic strength results in the formation of slower-sedimenting species which have not lost any significant amount of protein (see, for example, reference 1). Here we attempt to follow morphologically the process of magnesium removal from the 50S ribosomal subunit of Escherichia coli, using strain Q13 which exhibits a low RNase I activity. 50S particles, initially in 0.01M Tris, 0.0001M MgCl2, pH 7.4, were treated with EDTA either by dialysis against 0.002M Tris, 0.001M EDTA, pH 7.4, or by direct addition of a 0.1M solution of EDTA to a final concentration of 0.001M. On sucrose gradients made up in the Tris-EDTA buffer, the particles sedimented as a sharp peak in the 10-12S region, with no detectable slower - sedimenting species. Sucrose gradient analysis of the ribosomal RNA following EDTA treatment of 50S subunits showed that 23S RNA were largely converted to 16S, reflecting a low level of endogenous nuclease activity in the subunit preparation.

A Stable 44S Intermediate in the Autodigestion of 50S Ribosomal Subunits

1971 ◽  
Vol 29 ◽  
pp. 430-431
Author(s):  
John H. Nisbet ◽  
Henry S. Slayter
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Ribosomal Rna ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
Type Strains

Wild - type strains of Escherichia coli are known to contain as many as four endogenous nucleases (Ref. 1). These are commonly found associated with the ribosomes after extraction from the cell, but may be removed, with the exception of RNase IV, by washing the ribosomes in NH4Cl (at 0.2 M and higher concentrations). We have examined the effect of these nucleases on the 50S ribosomal subunit of one wild-type strain, K12 (Hfr 3000), by incubating the unwashed particles at 37° in the presence of varying magnesium concentrations.At 10-4 molar magnesium (slower at 10-3 molar), the 50S particle is converted to a species sedimenting at about 44S. About 20% of the total O.D260 is liberated at the same time. Continued incubation leads to the release of more O.D260 material while the RNA remaining in the 44S (Fig. 1) particle is progressively cleaved, eventually to the point where it consists of one principal fragment of molecular weight 0.42 x 106 daltons and several lesser fragments. The ribosomal RNA and proteins have been characterized by acrylamide gel electrophoresis.

scholarly journals Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

2017 ◽  
Vol 474 (2) ◽  
pp. 195-214 ◽  
Author(s):  
Salini Konikkat ◽  
John L. Woolford,
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Nucleolar Rnas ◽  
Large Ribosomal Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subunit Assembly ◽  
Nucleolar Rnas

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.

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