Proteins synthesized by rabbit reticulocyte membrane-bound ribosomes

1982 ◽  
Vol 60 (5) ◽  
pp. 580-585 ◽  
Author(s):  
Réal Lemieux ◽  
Claude Godin
Keyword(s):  
Protein Synthesis ◽  
Ionic Strength ◽  
Messenger Rna ◽  
Rna Degradation ◽  
Main Protein ◽  
Membrane Bound ◽  
Phenol Treatment

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact ribosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.

scholarly journals Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

1977 ◽  
Vol 168 (1) ◽  
pp. 1-8 ◽  
Author(s):  
J C Ramsey ◽  
W J Steele
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Size Structure ◽  
Large Fraction ◽  
Liver Homogenate ◽  
Structure And Function ◽  
Structural And Functional Properties ◽  
Membrane Bound ◽  
And Function

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

scholarly journals Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver

1982 ◽  
Vol 204 (1) ◽  
pp. 197-202 ◽  
Author(s):  
G Cairo ◽  
L Schiaffonati ◽  
M G Aletti ◽  
A Bernelli-Zazzera
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Messenger Rna ◽  
Relative Proportion ◽  
Liver Homogenate ◽  
Albumin Synthesis ◽  
Specific Proteins ◽  
Membrane Bound ◽  
Albumin Mrna

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

215 DISTRIBUTION OF DIMETHYLATION OF HISTONE H3K9 IS NOT AFFECTED BY DNA, MESSENGER RNA, AND PROTEIN SYNTHESIS IN PRONUCLEAR-STAGE PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 205
Author(s):  
K. E. Park ◽  
R. Cabot
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Control Group ◽  
Mrna Synthesis ◽  
Treatment Groups ◽  
Asymmetrical Distribution ◽  
Rna And Protein Synthesis ◽  
H3k9 Dimethylation ◽  
Differential Localization

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked with repression of transcription. Dimethylated H3K9 adopts a strict asymmetrical distribution in murine zygotes, with dimethylated H3K9 detectable only on maternally derived chromatin. In contrast, both male and female pronuclei in porcine zygotes can possess dimethylated H3K9; however, some asymmetry in H3K9 dimethylation exists between individual pronuclei, particularly in polyspermic embryos. The objective of this study was to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes. We hypothesized that the distribution of dimethylated H3K9 between individual pronuclei would not depend on alternations in chromatin structure induced by DNA or mRNA synthesis but would be affected by protein synthesis. To test this hypothesis, in vitro-matured porcine oocytes were fertilized in vitro, cultured in porcine zygote medium-3 containing 3 mg mL–1 of BSA, and allocated to 1 of 4 treatment groups: (1) incubation with 25 μg mL–1 of α-amanitin (α-AM), (2) incubation with 3 μg mL–1 of aphidicolin (APH), (3) incubation with 50 μg mL–1 of cycloheximide (CYC), and (4) nontreated controls. Embryos were removed from each treatment group at 10, 15, 20, and 25 h post gamete mixing, fixed, and processed to detect dimethylated H3K9 immunocytochemically. For monospermic embryos in the control group, 24% (7/29), 31% (8/26), 30% (7/24), and 20% (4/20) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the control group, 82% (32/39), 78% (31/40), 74% (28/38), and 65% (24/37) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the α-AM group, 29% (4/14), 14% (2/14), 8% (1/12), and 11% (1/9) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the α-AM group, 71% (15/21), 63% (12/19), 55% (10/18), and 47% (8/17) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the APH group, 31% (4/13), 23% (3/13), 23% (3/13), and 18% (2/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the APH group, 75% (15/20), 67% (12/18), 63% (12/19), and 56% (10/18) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For monospermic embryos in the CYC group, 33% (5/15), 25% (4/16), 14% (2/14), and 9% (1/11) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. For polyspermic embryos in the CYC group, 78% (18/23), 67% (16/24), 58% (14/24), and 59% (13/22) showed differential localization between pronuclei at 10, 15, 20, and 25 h, respectively. These results suggest that the distribution of dimethylated H3K9 between pronuclei is not affected by DNA, mRNA, or protein synthesis (P > 0.05), but is affected by the age of the pronuclei (P < 0.05).

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 146-157 ◽  
Author(s):  
D. D. Sabatini ◽  
G. Blobel
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Close Association ◽  
Membrane Integrity ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Cell Fractions

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

scholarly journals Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

1977 ◽  
Vol 74 (1) ◽  
pp. 43-57 ◽  
Author(s):  
MJ Grubman ◽  
JA Weinstein ◽  
DA Shafritz
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Cytosol Fraction ◽  
Initial Event ◽  
Glycoprotein G ◽  
Nonspecific Effects ◽  
Membrane Bound ◽  
Vesicular Stomatitis

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.

scholarly journals Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control

1992 ◽  
Vol 164 (1) ◽  
pp. 103-116 ◽  
Author(s):  
G. E. Hofmann ◽  
S. C. Hand
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Messenger Rna ◽  
Artemia Franciscana ◽  
In Vitro Translation ◽  
Translation Techniques ◽  
Polysome Profile ◽  
Anaerobic Dormancy ◽  
Qualitative Changes

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.

scholarly journals Identification of Small Molecule Inhibitors of Staphylococcus aureus RnpA

Antibiotics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 48 ◽  
Author(s):  
Jennifer M. Colquhoun ◽  
Lisha Ha ◽  
Andrew Beckley ◽  
Brinkley Meyers ◽  
Daniel P. Flaherty ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Small Molecule ◽  
Messenger Rna ◽  
Rna Degradation ◽  
Screening Assay ◽  
Whole Cell ◽  
Cellular Processes ◽  
Dual Functional ◽  
Worm Model

Staphylococcus aureus RnpA is thought to be a unique dual functional antimicrobial target that is required for two essential cellular processes, precursor tRNA processing and messenger RNA degradation. Herein, we used a previously described whole cell-based mupirocin synergy assay to screen members of a 53,000 compound small molecule diversity library and simultaneously enrich for agents with cellular RnpA inhibitory activity. A medicinal chemistry-based campaign was launched to generate a preliminary structure activity relationship and guide early optimization of two novel chemical classes of RnpA inhibitors identified, phenylcarbamoyl cyclic thiophene and piperidinecarboxamide. Representatives of each chemical class displayed potent anti-staphylococcal activity, limited the protein’s in vitro ptRNA processing and mRNA degradation activities, and exhibited favorable therapeutic indexes. The most potent piperidinecarboxamide RnpA inhibitor, JC2, displayed inhibition of cellular RnpA mRNA turnover, RnpA-depletion strain hypersusceptibility, and exhibited antimicrobial efficacy in a wax worm model of S. aureus infection. Taken together, these results establish that the whole cell screening assay used is amenable to identifying small molecule RnpA inhibitors within large chemical libraries and that the chemical classes identified here may represent progenitors of new classes of antimicrobials that target RnpA.

Sign in / Sign up

Export Citation Format

Share Document