Ribosome, preparation

Our first microcrystals were of Bacillus stearothermophilus SOS subunits and of 70S ribosomes from E. coli they were mainly grown from lower alcohols, toluene, or chloroform. Each ribosomal preparation required slightly different crystallization conditions, and often the preparation had almost been exhausted by the time conditions were optimised. We also found that crystals grew from active particles only. 

Cocucci, S. and Bagni, N., Polyamine-induced activation of protein synthesis in ribosomal preparation from Helianthus tuberosus tissue, Life Sci., 7, 113-120, 1968. 

With the aid of the electron microscope, ribosomes were first discovered as small, discrete, RNA-rlch particles in cells that secrete large amounts of protein. However, their role in protein synthesis was not recognized until reasonably pure ribosome preparations were obtained. In vitro radiolabeling experiments with such preparations showed that radioactive amino acids first were incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains. 

Figure 1.84 Schematic of translation. The mRNA codons are read and converted from nucleoside sequences to protein primary structure by means of cognate aminoacyl-tRNAs. All mRNA codons are translated at a ribosome (prepared from rRNA) that has two cognate aminoacyl-tRNA binding sites P (peptidyl) and A (aminoacyl). All tRNAs are "adaptors" that can bind a particular mRNA codon through their anticodon loop, using Watson-Crick base pairing, and also associate covalently with the appropriate amino acid residue coded for by the corresponding mRNA codon When two cognate aminoacyl-tRNA molecules bind mRNA in P and A sites (a), then both are close enough for peptide link formation to take place with the emergence of a peptide chain (b). As amino acyl tRNA molecules continue to dock sequentially onto mRNA codons (in the direction 5 (c), and amino acid residues continue to be added (W —> C ) (d),...

Reflect and Apply In the early days of research on protein synthesis, some scientists observed that their most highly purified ribosome preparations, containing almost exclusively single ribosomes, were less active than preparations that were less highly purified. Suggest an explanation for this observation. 

The less highly purified ribosome preparations contained polysomes, which are more active in protein synthesis than single ribosomes. 

Deuteration levels are dependent on the protonated carbon source in use for reason of the exact relationship that the carbon source has to the metabolic pathways in operation. Deuteration levels are best assayed by NMR methods [1-3], both in vivo and in vitro, and checked in the neutron experiment by a matchpoint determination [61]. For example, Escherichia coli grown in 100% H20 with glycerol will lead to 60% deuteration of RNA and 90% deuteration of protein. Of great practicality is the use of RNA and proteins which are deuterated to match out in exactly 100% H20. This can be done with E. coli grown on a glucose source in 76% H20 for RNA and 84% H20 for proteins. Ribosomes prepared from these are... 

Free- and membrane-bound ribosomes prepared from wheat germ have binding sites for concanavalin Four glycoproteins (mol. wts. 1.4-2.4X 10 ) were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in both classes of ribosomes. 

Neither ricin nor the toxic lectin modeccin are capable of inhibiting the elongation factor (EFl)-dependent binding of L-[ " C]phenylalanyl-tRNA although they are specific inhibitors of translocation. Inconsistencies in earlier reports may have arisen from an overestimation of the number of control ribosomes engaged in the binding reaction if trace amounts of EF2 contaminate the ribosomal preparations. 

Now let us consider the case of essential metal Ions that, nevertheless, produce deleterious effects in the wrong concentration. Magnesium Ions are required for protein synthesis, yet Mg " " Ions in too high concentration lead to errors, as is illustrated by the studies of Szer and Ochoa (16) on the incorporation of phenylalanine and leucine in a ribosomal preparation using poly(U) as the messenger RNA. UUC as well as the UUU codon in poly(U) code for phenylalanine, so that incorporation of the latter represents correct translation. UUA and UUG code for leucine, so that leucine incorporation in this system is "incorrect." At low Mg2+ concentration only phenylalanine is in fact incorporated. Phenylalanine incorporation is maximal at lOmM Mg " " as the Mg " " concentration is increased, however, leucine also becomes incorporated and its maximal incorporation is at 20mM Mg2+ ( ). 

Sixty-five per cent of the ribosome is ribonucleic acid, and the remainder is protein. The proteins associated with the ribosomal preparations are of two main types the genuine ribosomal and the newly synthesized polypeptide chains. 

If pure ribosomal preparation from E. coli is sedimented at a high Mg concentration, the preparation sediments giving a single peak sedimenting at 70 S. However, if Mg concentration is decreased, the same preparation sediments giving three peaks one at 70 S as before, another at 50 S and the third at 30 S (Figure 10.28). If Mg concentration is further reduced, the 70 S peak vanishes entirely and the 30 S and the 50 S peaks enclose more area. This tells us that the whole ribosome is actually formed of two subunits that can associate and dissociate depending on the Mg concentration. 

We are indebted to Mr. D. Haik for ribosome preparations. This work was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. 

Mean of triplicate assays of the transfer of leucine from sRNA into protein using ribosomes prepared from pooled thigh muscle from all rats in each experimental. group. 

Canavanine (oi-amino-y-guanidoxybutyric acid Fig. 1) and homo-aiginine are two naturally occurring analogues of arginine, present in some high plant families. They are incorporated into protein of various bacteria, as well as into rat-liver ribosomal preparations. 

Virus-specific phosphorylation of ribosomal proteins and/or proteins associated with ribosome preparations has been described (Ter-shak, 1978 James and Tershak, 1981) and thought to be a possible regulatory reaction involved in inhibition of cellular protein synthesis. Although no data are available to relate the phosphorylation events with the regulation of protein synthesis, it is interesting that one of... 

Tershak, D. A., 1978, Protein kinase activity of polysome-ribosome preparations from poliovirus-infected cells, Biochem. Biophys. Res. Commun. 80 283. 

In contrast to the preceding observations and conclusions, there are reports from one laboratory that the mechanism of peptide formation resembles that of protein synthesis. Cellfree preparations of B. hrevis were observed to synthesize gramicidin and tyrocidine provided that both a 140,000 X g supernatant solution and a ribosomal preparation are present (in addition to Mg+, ATP, phosphoenol-pyruvic acid, pyruvic kinase, glutathione, and amino acid mixture). The supernatant solution presumably supplies s-RNA and amino acid activating enzymes. In this system, peptide synthesis is suppressed by exposure of either the supernatant or ribosomal portions to RNase (Uemura et al., 1963)- Furthermore, neither chloramphenicol nor puromycin are capable of differentiating protein from peptide synthesis in this system the formation of each of the products is inhibited 98% by either 10 (xg/ml of the former or 100 fi.g/ml of the latter antibiotic (Okuda et al., 1964b). 

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