scholarly journals Reconstruction of the smaller subparticle of rabbit ribosomes from core-particle and split-protein fractions

1981 ◽  
Vol 194 (3) ◽  
pp. 931-939 ◽  
Author(s):  
R A Cox
Keyword(s):  
Protein Fraction ◽  
Protein Fractions ◽  
Particle Fraction ◽  
Core Particle ◽  
The Core ◽  
Diminished Capacity ◽  
Split Protein ◽  
Core Particles

The smaller subparticle of rabbit reticulocyte ribosomes was shown to yield core-particle and split-protein fractions on treatment with 2.5 M-NH4Cl/61 mM-MgCl2. The core-particle fraction was inactive in poly(U)-directed polyphenylalanine synthesis, but activity was restored after recombination with the split-protein fraction. Optimum ionic conditions for the reconstruction of active subparticles were found to be 0.75 M-NH4Cl/19 mM-MgCl2 at 0 degrees C. Improved extents of reconstruction were obtained when the core-particles were isolated by methods that avoided pelleting. Core-particles isolated from subparticles pretreated with either proteinases or ribonucleases had diminished capacity to become re-activated.

scholarly journals Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction

1976 ◽  
Vol 160 (3) ◽  
pp. 533-546 ◽  
Author(s):  
R A Cox ◽  
P Greenwell
Keyword(s):  
Protein Fraction ◽  
Protein Fractions ◽  
Molar Ratio ◽  
Core Particle ◽  
Original Activity ◽  
Two Dimensional Gel Electrophoresis ◽  
The Core ◽  
Split Protein ◽  
Synthesis Activity ◽  
Protein Moiety

We report the reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69mM-MgCl2 at 0° C. This treatment led to the loss of about eight proteins (approx. 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by two-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly (U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4° C against 0.3M-NH4Cl/15mM-MgCl2/1mM-dithiothreitol/15% (v/v) glycerol/20mM-Tris/HCl, pH 7.6, and then heated for 1 h at 37° C. The recovery was 40-80% of the original activity. Raising the concentration of MgCL2 to 300 mM in 2.5 M-NH4CL led to the removal of seven rather than eight proteins, and the core particle remained active in the puromycin reaction. We infer that the protein retained by raising the concentration of Mg2+ is an essential component of the peptidyltransferase centre of the ribosome.

scholarly journals Repositioning septins within the core particle

2019 ◽  
Author(s):  
Deborah C. Mendonça ◽  
Joci N. Macedo ◽  
Rosangela Itri ◽  
Samuel L. Guimaraes ◽  
Fernando L. Barroso da Silva ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Self Assembly ◽  
Binding Proteins ◽  
Structural Information ◽  
Core Particle ◽  
Affinity Tag ◽  
The Core ◽  
Gtp Binding ◽  
True Structure ◽  
Core Particles

AbstractSeptins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

The core particles of herpes simplex virus

1974 ◽  
Vol 20 (5) ◽  
pp. 731-734
Author(s):  
Claude J. Henry
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Core Particle ◽  
Particle Assembly ◽  
Thin Sections ◽  
The Core ◽  
Infected Cells ◽  
Simplex Virus ◽  
Outer Capsid ◽  
Core Particles

Numerous core particles measuring 45–50 nm in diameter were observed in the nuclei of cells infected with herpes simplex virus for 20 h. The sites for core-particle assembly appeared to be closely associated with viral DNA-replicating areas in the nucleus. The core particles were subsequently observed to be associated with the protein subunits that comprise the outer capsid of the virus. Outer capsids were formed only in areas in which core particles were present; however, numerous empty capsids (lacking a core particle) measuring 80–90 nm in diameter were also observed. The effect of trypsin digestion on thin sections of infected cells revealed that the core particles were selectively degraded by trypsin, but that the outer capsid and envelope remained relatively intact. The core particles appeared to be more sensitive to trypsin treatment after acquisition of a capsid and envelope.

scholarly journals Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins

1980 ◽  
Vol 186 (3) ◽  
pp. 861-872 ◽  
Author(s):  
R A Cox ◽  
P Greenwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Fractions ◽  
Molecular Architecture ◽  
Reductive Methylation ◽  
Active Centre ◽  
Split Protein ◽  
Cytoplasmic Ribosomes ◽  
Core Particles

It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.

scholarly journals Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases

1980 ◽  
Vol 190 (1) ◽  
pp. 199-214 ◽  
Author(s):  
R A Cox ◽  
S Kotecha
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
High Salt ◽  
Protein Fractions ◽  
Marginal Effect ◽  
Trypsin Treatment ◽  
Salt Shock ◽  
Split Protein ◽  
Core Particles

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16–100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

scholarly journals nanocrystals (0 ≤ x ≤ 1): growth, size control and shell formation on β-NaCeF4:Tb core particles

CrystEngComm ◽  
2020 ◽  
Vol 22 (46) ◽  
pp. 8036-8044
Author(s):  
Jannis Wehmeier ◽  
Markus Haase
Keyword(s):  
Size Control ◽  
Lattice Parameters ◽  
Core Material ◽  
Shell Formation ◽  
Shell Material ◽  
Strontium Content ◽  
The Core ◽  
Lighter Lanthanides ◽  
Core Particles

is an interesting shell material for β-NaREF4 particles of the lighter lanthanides (RE = Ce, Pr, Nd), as variation of its strontium content x allows to vary its lattice parameters and match those of the core material.

scholarly journals Crystallization and Preliminary X-ray Analysis of the Core Particle of Bluetongue Virus

Virology ◽  
1995 ◽  
Vol 210 (1) ◽  
pp. 217-220 ◽  
Author(s):  
J.N. Burroughs ◽  
J.M. Grimes ◽  
P.P.C. Mertens ◽  
D.I. Stuart
Keyword(s):  
Bluetongue Virus ◽  
Core Particle ◽  
X Ray ◽  
The Core

Influence of Lysozyme on the Biomimetic Growth of Silica Tubes in Porous Membranes

2007 ◽  
Vol 1008 ◽  
Author(s):  
Clémentine Gautier ◽  
Rémi Courson ◽  
Pascal Jean Lopez ◽  
Jacques Livage ◽  
Thibaud Coradin
Keyword(s):  
Porous Membranes ◽  
Shell Formation ◽  
Core Particle ◽  
Biomimetic Models ◽  
Membrane Pores ◽  
The Core ◽  
Core Shell Structure ◽  
Poly Carbonate ◽  
Tube Shell

AbstractPore channels of poly-carbonate membranes were recently used as biomimetic models to study the effect of confinement on silicate condensation, leading to the formation of silica tubes exhibiting a core-shell structure. In this work, we pre-immobilized lysozyme on the membrane pores, inducing the modification of the tube shell formation process, and variation in core particle size. These data strengthen previous assumptions on the role of interfacial interactions on the growth of the tube shell and indicate that such interactions also influence the core particle formation. Such approach therefore seems suitable to mimic the formation of silica/protein multilayers as found in several biomineralizing organisms

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