Aminoacyl-transfer RNA

Figure 8.4 outlines the proeess of protein synthesis involving the ribosome, ruRNA, a series of aminoacyl transfer RNA (tRNA) moleeules (at least one for eaeh amino aeid)... 

Although all tetracyclines have a similar mechanism of action, they have different chemical structures and are produced by different species of Streptomyces. In addition, structural analogues of these compounds have been synthesized to improve pharmacokinetic properties and antimicrobial activity. While several biological processes in the bacterial cells are modified by the tetracyclines, their primary mode of action is inhibition of protein synthesis. Tetracyclines bind to the SOS ribosome and thereby prevent the binding of aminoacyl transfer RNA (tRNA) to the A site (acceptor site) on the 50S ri-bosomal unit. The tetracyclines affect both eukaryotic and prokaryotic cells but are selectively toxic for bacteria, because they readily penetrate microbial membranes and accumulate in the cytoplasm through an energy-dependent tetracycline transport system that is absent from mammalian cells. 

The translation of the mRNA into proteins is the final step in the biological flow of information (see Fig. 6.1). Similar to other macromolecular polymerizations, protein synthesis can be divided into initiation, chain elongation, and termination. Critical players in this process are the aminoacyl transfer RNAs (tRNAs). These molecules form the interface between the mRNA and the growing polypeptide. Activation of tRNA involves the addition of an amino acid to its acceptor stem, a reaction catalyzed by an aminoacyl-tRNA synthetase. Each aminoacyl-tRNA synthetase is highly specific for one amino acid and its corresponding tRNA molecule. The anticodon loop of each aminoacyl-tRNA interacts... 

The answer is b. (Hardman, p 1131.) Chloramphenicol inhibits protein synthesis in bacteria and, to a lesser extent, in eukaryotic cells. The drug binds reversibly to the SOS ribosomal subunit and prevents attachment of aminoacyl-transfer RNA (tRNA) to its binding site. The amino acid substrate is unavailable for peptidyl transferase and peptide bond formation. 

Brown JR, Gentry D, Becker JA, Ingraham K, Holmes DJ, Stanhope MJ (2003) Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens. EMBO Rep 4 692-698... 

Aminoacyl-Transfer-RNA Synthetases Read the Genetic Code... 

The small ribosomal subunit is loaded with initiation factors, messenger RNA, and initiation aminoacyl-transfer RNA. 

Minocycline, a tetracycline, is indicated in syphilis or gonorrhea in patients sensitive to penicillin. In addition, it may be used in uncomplicated urethral, endocervical, or rectal infection, and in uncomplicated gonococcal urethritis in men (see also Figure 96). Tetracyclines enter bacterial cells by both passive diffusion and active transport, and then accumulate intraceUularly. This does not occur in mammalian cells. The tetracyclines bind to the 308 subunit of the bacterial ribosome in such a way that the binding of the aminoacyl-transfer RNA to the acceptor site on the messenger RNA ribosome complex is blocked (see Figure 96). 

The genetic code (which includes the codon) serves as a basis for establishing how genes encoded in DNA are decoded into proteins. A critical interaction in protein synthesis is the interaction between the codon in messenger RNA (mRNA) and the anticodon in an aminoacyl-transfer RNA (aminoacyl-tRNA). 

Ono, Y., Skoultchi, A., Waterson, J., and Lengyel, P. (1969) Peptide chain elongation GTP cleavage catalysed by factors binding aminoacyl-transfer RNA to the ribosome. Nature, 222, 645-648. 

As reviewed by Stulberg and Novelli in 1962, several methods are available to measure the activity of the aminoacyl-RNA synthetases. The kinetic parameters are generally derived using the ATP exchange reaction, and the isolation of the aminoacyl-transfer RNAs. 

Puromycin inhibits protein synthesis in numerous cellular species (including eukaryotic cells). It was shown, by studies in subcellular systems for protein synthesis, that this antibiotic hinders the amino-acid transfer from the aminoacyl-transfer-RNA, to the growing polypeptide chains on the ribosomes. In 1959, Yarmolinsky and de la Haba postulated that puromycin acts as an aminoacyl-transfer-RNA analogue, because of its structural resemblance with the amino acid carrying acceptor extremity of an aminoacyl-transfer RNA (Fig. 1). This hypothesis is now completely confirmed. Puromycin induces the release of unfinished polypeptide chains, when acting in a protein-synthesizing system, and these polypeptides... 

Numerous experiments demonstrate that tetracyclines mainly act by impairing the fixation of aminoacyl-transfer RNA, to the complex ribosome-messenger RNA (absolute prerequisite for peptide-bond formation). However, chlortetracycline also inhibits peptide-bond formation between puromycin and the peptidyl-transfer-RNA molecule already bound to the ribosome (Cemi et al., 1969). 

Chloramphenicol does not inhibit messenger-RNA fixation to the 30 S subunit, nor aminoacyl-transfer RNA binding to the messenger-RNA-ribosome complex. The precise mode of action remains uncertain (see also Para. 8.1.8.1.S). 

These antibiotics act at the level of the SOS ribosomal subunit, and do not seem to inhibit aminoacyl-transfer-RNA fixation to the ribosomes. 

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. 

According to the mechanism of translation outlined in the introduction, it is clear that analogues are activated before being incorporated into protein, and compete with the protein amino acid for the aminoacyl-transfer-RNA synthetases. They are afterwards transferred to specific transfer RNAs. When the natural amino-acid analogue has been transferred to the transfer-RNA molecule, it takes no part in determining the specificity of polypeptide synthesis. The incorporation of Ala in place of Cys, after the chemical reduction of Cys-tRNA has been outlined before (see Para. 8.1.2). Similarly Cys-tRNA can be mildly oxidized to CysSOsH-tRNA thereafter cysteine sulfonic acid becomes incorporated into protein. This appears to be a suitable procedure to incorporate an analogue which per se is not specific enough to be incorporated (CysSOsH itself is not incorporated). Such a procedure is, however, essentially restricted to in vitro studies. 

The first step in the translation process is the activation of the amino add into an aminoacyl adenylate mediated by a spedfic aminoacyl-transfer-RNA synthetase stage, followed by the linkage of the amino add to the low molecular weight transfer RNAs (see Sect B, Para. 8.1.2). 

However, formation of the specific repressor by the regulator gene is evidently only one of the possible methods of repression of transcription of operons. Other methods of repression of the structural genes in bacteria have recently been found (Richmond, 1967 Bretscher, 1968) and the repressor function of aminoacyl-transfer RNAs in repression of the enzymes of amino acid synthesis has been studied in more detail (Freundlich, 1967). 

Because of safety resistance and life-threatening adverse effects, particularly bone-marrow aplasia, it is now just used in the tropical treatment and in some countries because of its low price. Within the ceU, it binds to the SOS subunit of the bacterial ribosome and inhibits bacterial protein synthesis by preventing attachment of aminoacyl transfer RNA to its acceptor site on the ribosome. ... 

Bosshard, H. R., 1976, Theories of enzyme specificity and their application to proteases and aminoacyl-transfer RNA synthetases, Experientia 32 949. 

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