Breakdown and Reassembly of Rat Liver Ergosomes after Administration of Ethionine or Puromycin (original) (raw)
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Dissociation of Single Ribosomes as a Preliminary Step for Their Participation in Protein Synthesis
European Journal of Biochemistry, 1973
32P-labelled derived 40-S ribosomal subunits and 80-S monoribosomes entered the polyribosome fraction of a rabbit-reticulocyte lysate active in protein synthesis by a process that was dependent on time, energy and temperature. Both 40-S and 60-S subparticles from the labelled monoribosome entered the polyribosomes a t the same rate. The addition of excess unlabelled 40-S subparticles to the cell-free system containing labelled monoribosomes increased the ratio of 6 0 3 to 40-S-subparticle radioactivity in the polyribosome fraction. This indicated a competition between added unlabelled and monoribosome-derived 40-5 subparticles for entry into polyribosomes. From the evaluation of the translation time of mRNA, of the rate of monoribosomepolyribosome exchange, and of the equilibrium distribution of the cycling labelled monoribosomes and unlabelled subparticles with polyribosomes it is concluded that the dissociation of single ribosomes into subunits is an obligatory step for their entry into the poIyribosoma1 fraction and that this process is coupled with initiation of mRNA translation.
Ribosomes and protein synthesis
2016
Let us start at the very beginning. Between 1897 and 1899, G. Gamier, in France, published elegant microscope studies describing a basophilic component ofthe cytoplasm of glandular cells (1). Because of what he thought its role might be in the elaboration and transformation of secretory products, he gave a Greek name to these concepts-ergastoplasm (work plasm). Gamier's research was extended by others-particularly A. Prenant, R. R. Bensley, and A. Matthews-to include other cell types, so that by the early part of this century ergastoplasm came to be a generally accepted term for a specific basophilic area of the cytoplasm. These early studies are extensively reviewed by F. Haguenau (1). The next major advance was to show that basophilia was due to RNA: in 1933, J. Brachet used RNase (2) ; in 1939, T. Caspersson used ultraviolet spectrophotometry (3); in 1943, J. N. Davidson and C. Waymouth used chemical methods (4). The high correlation which was shown between the amount ofRNA in various cells and the postulated protein-synthesizing capacity of those cells led Caspersson in 1941 (5) and Brachet in 1942 (6) to proclaim the importance of RNA in the process of protein synthesis. As can be imagined, this conjecture spurred many scientists in the next decade to try to answerthree questions. In what form was this cytoplasmic RNA? Did it really have a role in protein synthesis? Ifso, what was the role? Various methods were used : extraction and chemical procedures; extraction and physicochemical procedures, such as ultracentrifugation; and, because electron microscopy was becoming more and more refined, visualization. We now know that the RNA is in the form of ribosomes, and that the proteins of the ribosomes are involved in the many individual steps of protein synthesis; however, the function of ribosomal RNA is still elusive. The intensity of the research in the 1940s is caught very well in Haguenau's chapter on the visualization aspect (1) and in Magasanik's 1955 monograph (7) on the extraction and chemical properties of what were then called "pentose nucleoprotein." Confusion abounded, in good part due to the terminologies developed for the different techniques, such as Gamier's ergastoplasm, K. Porter's "endoplasmic reticulum" (8), A. Claude's "microsome" fraction (9), G. Palade's "small particulate component" (10), and the "nucleoprotein" preparations or particles discovered by various workers. The last are re
Messenger-Rna Attachment to Active Ribosomes
Proceedings of the National Academy of Sciences, 1962
In a current experiment, we have found that a protein fraction isolated from the 105,000 X g supernatant can catalyze a similar RNA synthesis in the presence of four nucleoside triphosphates and the E. coli DNA prepared by the method of Marmur (J. Mol. Biol., 3, 208, 1961). In this system also, the addition of salmine enhances the initial rate of the synthesis and prevents the degradation of the product. The rate is about comparable to that of the crude system. However the reaction practically ceases after ten minutes even if the nucleoside triphosphates are further added at 10-min intervals. The ratio of the synthesized RNA to the DNA added is around 0.5. The result might be explained by assuming a factor necessary for the liberation of the synthesized RNA from the DNA. In the crude system, the RNA would be continuously liberated from DNA in the presence of the "factor;" in the "purified" system, the RNA would not be liberated because of the absence of the "factor."
Isolation of ribosomes and assay systems for testing the translational apparatus have been part of the experimental routine of many laboratories for more than two decades, and excellent collections of the methods have been published previously, including two books in this series. However, a number of methods have been gradually and continuously improved and optimized. Major developments have included the improvement of ribosome isolation procedures, the establishment of highly efficient assay systems, and the application of heteropolymeric mRNA. The protocols in this chapter are concerned with the isolation of polysomes, ribosomes, and ribosomal subunits from prokaryotic and eukaryotic sources, as well as test systems for both total protein synthesis and single ribosomal functions, taking into account the recent developments.