Active polysomes

HMG-CoA reductase is also subject to translational control by a mevalonate-derived non-sterol regulator (D. Peffley, 1985 M. Nakanishi, 1988). Tliis component of the regulatory mechanism can be observed only when cultured cells are acutely incubated with statins, which block mevalonate formation. Under those conditions, sterols have no effect on HMG-CoA reductase mRNA translation however, mevalonate reduces the HMG-CoA mRNA translation by 80% with no change in mRNA levels. Translational control of hepatic HMG-CoA reductase by dietary cholesterol was shown in an animal model in which polysome-associated HMG-CoA reductase mRNA was analyzed in cholesterol-fed rats (C.M. Chambers, 1997). It was found that cholesterol feeding increased the portion of mRNA associated with translationally inactive monosomes and decreased the portion of mRNA associated with translationally active polysomes. The mechanism of HMG-CoA reductase translational control has not been elucidated. 

It is noteworthy that free mRNP particles are a temporary untranslatable form of mRNA in the cytoplasm and that proteins associated with the active polysomal mRNP or the repressed mRNP are different [6, 7]. There is now some evidence that ribo-nucleoprotein particles are dynamic structures and that protein exchanges occur between the cytoplasmic mRNA-associated proteins and free proteins. Involvement of mRNA-associated proteins in the regulation of protein synthesis has been considered [7-10] and post-translational modification of these proteins as a regulatory mechanism might be considered. 

The template properties of virus RNA are manifested, as in the case of messenger RNA of the host cell, during its interaction with ribosomes. Just as in the case of normal intracellular synthesis, polysome structures of the type illustrated in Fig. 8 are formed under these circumstances. The formation of active polysomes during interaction between virus RNA and cell ribosomes have been studied in particular detail in the case of poliovirus infections of HeLa cell cultures (Penman et al., 1963 Scharff et al., 1963 Rich et al., 1963). In this condition virus-specific polysomes are formed which are much bigger than the polysomes of uninfected HeLa cells, as a result of the polycistron character of virus RNA. Some of them contained as many as 60 ribosomes. 

Demonstration of active polysomes in the unfertilized egg. These experiments have also shown that incorporation of amino acids into protein by unfertilized eggs occurs in association with polysomes, as it does in fertilized eggs. Measurements of optical density at 260 m/i show the presence of RNA-containing material in the polysome region... 

Bell, E., Humphreys, T., Slayter, H. S., and Hall, C. E. (1965). Configuration of inactive and active polysomes of the developing down feather. Science 148, 1739-1741. 

A ribosome is a cytoplasmic nucleoprotein stmcture that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. 

We utilize three independent methods to assess a compound s effect in vivo on translation—metabolic 35S-methionine/cysteine labeling, polysome profile analysis, and effects on transfected reporter gene activity. 

Alternatively, poor efficiencies of inhibitor mRNAs may be due to their Incorporation into rlbonucleoproteln particles (RNPs) such as found in sea urchin embryos (21). Newly made mRNA in the embryos is found in RNPs and they apparently have "weak" template activities while in these particles. The presence of newly synthesized tomato mRNA in similar particles might explain the apparently low translational efficiencies noted herein. The use of chaotropic buffers in the preparation of tomato leaf mRNA (11) would not differentiate between free or polysome-bound mRNAs and those complexed in RNPs. If an RNP or similar particle is involved, then its role must be a temporal one since a second wound does not repeat the phenomenon (Fig. 4). 

Inside the cell, aminoglycosides bind to specific 30S-subunit ribosomal proteins (S12 in the case of streptomycin). Protein synthesis is inhibited by aminoglycosides in at least three ways (Figure 45-3) (1) interference with the initiation complex of peptide formation (2) misreading of mRNA, which causes incorporation of incorrect amino acids into the peptide and results in a nonfunctional or toxic protein and (3) breakup of polysomes into nonfunctional monosomes. These activities occur more or less simultaneously, and the overall effect is irreversible and lethal for the cell. 

Protein synthesis can be carried out by ribosomes free in the cytosol. In eukaryotes, ribosomes also carry out protein synthesis while bound to the surface of the endoplasmic reticulum. In addition, a given mRNA molecule usually has more than one active ribosome translating it into protein an assembly of several ribosomes on a single mRNA is called a polyribosome, or polysome for short. 

Rapid Translation of a Single Message by Polysomes Large clusters of 10 to 100 ribosomes that are very active in protein synthesis can be isolated from both eukaryotic and bacterial cells. Electron micrographs show a liber between adjacent ribosomes in the cluster, which is called a polysome (Fig. 27-27). The connecting strand... 

Probes may also consist of DNA copied from mRNA. This is known as cDNA and is also widely used to determine indirectly the sequences of mRNA molecules. Messenger RNA may be isolated from the total cellular RNA by affinity chromatography on bound poly (dT) or poly (U). These materials selectively hold RNA with the poly (A) tails characteristic of most eukaryotic mRNA (see Chapter 28). Another source of mRNA is polyribosomes (polysomes), which are "reading" mRNA and actively making proteins. 

OH group. (2) it must recognize the proper triplet on the messenger RNA-ribosome aggregate. Having these properties, the transfer RNA accepts or forms an intermediate transfer RNA-aminn acid thal finds its way to the polysome, complexes at a triplet coding for the activated amino acid, and allows transfer of the amino acid into peptide linkage. 

Polyribosome (polysome). A complex of an mRNA and two or more ribosomes actively engaged in protein synthesis. 

Translating ribosomes in eukaryotes are located in different places in the cell depending on the fate of their proteins. Free polysomes are in the cytoplasm and synthesize cytoplasmic proteins and those that are bound for most intracellular organelles, for example, the nucleus. Members of the second class of polysomes, membrane-bound polysomes, are attached to the endoplasmic reticulum (forming the rough ER), and synthesize exported proteins. In cells that are actively secreting enzymes or hormones (for example, those in the pancreas), most of the protein synthesis occurs on the rough ER. 

The reactions of phase 2 relate to the attachment of the bridge-carbohydrate residues to the polypeptide chain. There is evidence showing that this addition occurs while the polypeptide chain is still attached to, or perhaps still being synthesized on, the ribosomes.101-103 Thus, 14C-labeled 2-amino-2-deoxy-D-glucose, injected into the circulatory system of the rat, was incorporated into protein in the ribosomes of the rough endoplasmic-reticulum of the liver. Administration of puromycin caused release of the 14C-labeled glycoprotein, which could be isolated by acid-precipitation methods. Examination of the radioactivity data revealed that the subcellular structures most actively involved in glycoprotein synthesis were the ribosomes bound to the membrane, and not free polysomes. 

The mechanism of N-acetylation of a-crystallin is quite interesting. The N-terminal residue has been identified as N-acetyl methionine. This methionine residue is derived from Met-tRNA et which is responsible for the initiation of the polypeptide chain and not Met-tRNAmet which incorporates the methionine residue in the growing polypeptide chain. It is clear that the N-acetylation is a true posttranslational process since acetyl Met-tRNA cannot replace formyl Met-tRNA et. Moreover, N-acetylation occurs when the polypeptide chain has reached a length consisting of approximately 25 amino acid residues. Other proteins, such as ovalbumin, are also acetylated during the early stages of polymerization on the polysome, and the protein acetyltransferase activity must therefore be associated with the protein-synthesizing apparatus. 

A rapid translational response of ferritin has been reported to occur after ferritin mRNA was recruited to polysomes [14]. An increase in the cytosolic ferritin mRNA and ferritin protein following ischemia and reperfiision of the intestine was also reported [26]. Reperfusion has been shown to cause ferritin degradation, followed by activation of ferritin synthesis [27,28]. 

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