Protein synthesis in cell-free systems

Peptide synthesis is a preparative technique as, to some extent, is protein synthesis in cell-free systems (Bodansky and Bodansky 1994 Pennington and Dunn 1994 ... 

The inhibitory effect of ricin on protein synthesis in cell-free systems is greatly increased in the presence of / -mercaptoethanol. This is because free A chain is the enzymatically active toxin, whereas A chain linked to B chain in whole ricin is not active [ 121]. In the absence of reducing agents, even high concentrations of intact toxin do not inactivate ribosomes. Presumably the catalytic site on the A chain is formed or exposed only when the A chain is released from the B chain. Free A chain, however, is non-toxic to intact cells since, in the absence of B chain, it lacks the ability to bind to and enter cells. Ricin A-chain preparations frequently show some level of toxicity, however, because the complete removal of contaminating B chain can be difficult to achieve [ 122]. 

Also, we do not yet know how viruses such as YSY, which have capped messengers, inhibit cellular protein synthesis. Whatever the strategy that YSY uses to turn off the host, poliovirus is apparently resistent to this step, since poliovirus grows in YSY infected cells. If YSY does inactivate an initiation factor other than eIF-4B, then this would suggest that poliovirus mENA translation by-passes the requirement for several cellular initiation factors. This finding emphasizes our lack of knowledge of the exact initiation factor requirement for viral and cellular protein synthesis in cell-free systems. 

A controversy has arisen about the participation of the mitochondria in thyroxine s stimulation of protein synthesis in cell-free systems. Whereas some investigators claimed to have observed the effect in microsomal preparations completely devoid of mitochondria, others claim that the presence of mitochondria in the cell-free preparation is indispensable to stimulation. The mitochondria do not act simply by generating ATP because their replacement by a classical ATP-generating system does not contribute to the stimulation of protein synthesis in microsomes of thyroidectomized animals injected with thyroxine. 

Coppola, G., and Bablanian, R., 1983, Discriminatory inhibition of protein synthesis in cell-free systems by vaccinia transcripts, Proc. Natl. Acad. Sci. USA 80 75. 

It is well known that synthetic or natural dsRNAs can serve as potent inhibitors of protein synthesis in various in vitro translation systems derived from mammalian cells (Ehrenfeld and Hunt, 1971 Kaempfer and Kaufman, 1973). A variety of other viral dsRNAs inhibit cell-free protein synthesis. The presence of dsRNA has also been detected by crosslinking experiments in HeLa cells infected with en-cephalomyocarditis virus or the t G114(I) mutant of VSV (Nilsen et al., 1981). Reovirus mRNA and polyadenylated vaccinia RNA transcribed in vitro have both been shown to contain dsRNAs which inhibited protein synthesis in cell-free systems (Baglioni et al., 1978 McDowell et al., 1972). Studies such as these with viruses other than rhabdoviruses are discussed in other chapters of this volume. The question that must be kept in mind is whether the in vitro inhibition of translation initiation by VSV dsRNA or any other viral dsRNA truly represents the sequence of events that takes place in the virus-infected cell. 

Kigawa, T., Matsuda, T., Yabuki, T., and Yokoyama, S. (2008) Bacterial cell-free system for highly efficient protein synthesis, in Cell-Free Protein Synthesis (eds A.S. Spirin and J. R. Swartz), Wiley-VCH Verlag GmbH, Weinheim, Germany, pp. 83-97. 

Although interferons are mediators of immune response, different mechanisms for the antiviral action of interferon have been proposed. Interferon-a possesses broad-spectrum antiviral activity and acts on virus-infected cells by binding to specific cell surface receptors. It inhibits the transcription and translation of mRNA into viral nuoleic acid and protein. Studies in cell-free systems have shown that the addition of adenosine triphosphate and double-stranded RNA to extracts of interferon-treated cells activates cellular RNA proteins and a oellular endonuclease. This activation causes the formation of translation inhibitory protein, which terminates production of viral enzyme, nucleic acid, and structural proteins (28). Interferon also may act by blocking synthesis of a cleaving enzyme required for viral release. 

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

GTP is also essential for polypeptide synthesis in cell-free systems. The mechanism for the GTP utilization in protein synthesis is not yet clear, although GTP may supply the energy needed for the coordinated reactions involving the binding of amino acyl-tRNA, formation of the peptide bond and the movement of messenger RNA along ribosomes. 

In contrast to these results, the process of tyrocidine and gramicidin biosynthesis has been compared to protein synthesis, but this evidence was obtained in cell-free systems of B. brevis . Yet a difference in the mechanism of protein and antibiotic synthesis has also been reported for gramicidin polymyxin and mycobacillin and seems now to be... 

Ozawa K, Jergic S, Crowther JA, Thompson PR, Wijffels G, Otting G, Dixon NA (2005) Cell-free protein synthesis in an autoinduction system for NMR studies of protein-protein interactions. J Biomol NMR 32 235-241... 

Compared with some of the other mycotoxins such as aflatoxin, the trichothecenes do not appear to require metabolic activation to exert their biological activity.50 After direct dermal application or oral ingestion, the trichothecene mycotoxins can cause rapid irritation to the skin or intestinal mucosa. In cell-free systems or single cells in culture, these mycotoxins cause a rapid inhibition of protein synthesis and polyribosomal disaggregation.35 47 50 Thus, we can postulate that the trichothecene mycotoxins have molecular capability of direct reaction with cellular components. Despite this direct effect, it is possible to measure the toxicokinetics and the metabolism of the trichothecene mycotoxins. 

Although it has been generally assiimed that the effects of haemin on protein synthesis might be particular to reticulocytes, recent work has shown that haemin, GTP and sugar phosphates stdLmulate initiation in cell-free systems from other cells in a... 

In an attempt to pinpoint the site of action of the thyroid hormone in the pathway for protein synthesis, incorporation of labeled amino acid in cell-free systems of thyroidectomized animals was compared to that in similar systems of thyroidectomized animals injected with thyroxine. Thyroidectomy slowed amino... 

Indeed, poly (U-G) and poly (U-Aza G) direct the incorporation of the same amino adds (the spedfidty is not altered) into polypeptides, but poly (U-Aza G) is less efficient when added to a subcellular protein-synthesis system derived from E. coli. With codons containing two Aza G, the interaction between the amino-acyl-transfer RNA and the triplet is so weakened that the translation may be interrupted, giving incompl polypeptide chains, which remain attached to the ribosomes. Ibis has been demonstrated in cell-free systems derived from aza-guanine-treated B. cereus. This also explains the differences observed by Chantreime between the d ree of inhibition of global protein synthesis and that of spedfic enzymes. 

In cell-free systems the inhibition of the transfer of aminoacyl-transfer RNAs to polypeptide (at the ribosome level) is probably the primary effect The most interesting effect of cycloheximide is that protein synthesis by isolated mitochondria of eukaryotic cells, like bacterial ribosomes, but unlike mammalian and yeast cytoplasmic ribosomes, is not inhibited over a wide range of concentrations. Despite this selective action, cycloheximide is extremely harmful to the biogenesis of mitochondria in vivo, due to a large contribution of the microsomal protein synthesizing system in the formation of mitochondrial proteins. 

As shown in Table I, there is substantial in vitro evidence that GTP stimulates anabolic processes in cell-free systems ranging from nucleic acid and protein synthesis through nucleic acid function, protein glyco-sylation, assembly, phospholipid biosynthesis, and even cell wall biosynthesis. As summarized in Table I, GTP influences on 12 different anabolic processes have been characterized in cell-free systems. In each case, GTP stimulates the anabolic process involved. 

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