Aminoacyl, transfer

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. 

Most naturally occurring ribozymes catalyze phosphoryl transfer reactions, where a sugar 2 -OH or 3 -OH attacks a phosphodiester linkage [121]. The two main classes are intramolecular ribozymes, where the sugar-OH nucleophile attacks its own 3 -phosphodiester (Figure 5.21a) and intermolecular ribozymes, where the nucleophile comes from a different RNA strand (Figure 5.21b). There is also evidence that the RNA catalyzes the peptide bond-forming aminoacyl transfer reaction in ribosomes [124,125]. 

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... 

The aminoacyl transfer reaction, one of the latter stages in protein synthesis, involves incorporation of amino acids from soluble ribonucleic acid-amino acid into ribosomal protein. This reaction requires guanosine triphosphate and a soluble portion of the cell. Evidence has been obtained with rat liver preparations that aminoacyl transfer is catalyzed by two protein factors, aminoacyl transferases (or polymerases) I and n, which have been resolved and partially purified from the soluble fraction. Transferase n activity has also been obtained from deoxycholate-soluble extracts of microsomes. With purified transferases I and n, incorporation is observed with relatively low levels of GTP its sulfhy-dryl requirement is met by a variety of compounds. The characteristics of this purified amino acid incorporating system, in terms of dependency on the concentration of its components, are described. 

Studies in this laboratory for the past several years have been concerned with the elucidation of the latter steps in this series of reactions. Specifically, efforts have been directed toward the characterization of the reaction involving the transfer of aminoacyl sRNA to mammalian ribonucleoprotein particles, the enzymatic and cofactor requirements, and possible intermediates in this process. The evidence obtained indicates that aminoacyl transfer is an enzymatic reaction requiring at least two enzyme fractions, which have been resolved and partially purified, GTP and a sulfhydryl compound further, the possibility exists that a ribosome-bound sRNA-amino acid (or peptide) compound is formed as an intermediate in this reaction. 

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

Table I. Aminoacyl Transfer to Microsomes with Crude and Purified Transferring Preparations...

Studies with ribosomes indicated that, in contrast to experiments with microsomes, the purified transferring enzyme described above failed to catalyze aminoacyl transfer (Tablem) however, the crude pH 5 Supernatant was active with both particle preparations. As described in Table IV, when the incubations with purified enzyme (transferase I) were supplemented with the dialyzed deoxycholate-soluble microsomal extract (transferase II) obtained during the isolation of ribosomes (Figure 1), transferring activity was restored (6, 7). Glutathione was also... 

Table III. Aminoacyl Transfer to Intact Rat Liver Microsomes and to Ribosomes...

Table IV. Effect of Soluble and Microsomal Extracts on Aminoacyl Transfer...

Since pH 5 Supernatant was active in the transfer of sRNA-bound amino acids, it suggested that both of the essential fractions described above were also present in this crude soluble preparation. Experimental verification of this suggestion is presented in Table V (8). Fractionation of the pH 5 Supernatant with ammonium sulfate yielded two fractions, 0 to 35% A.S. residue and 35 to 60% A.S. residue, which by themselves catalyzed aminoacyl transfer in the presence of glutathione. Reprecipitation of the 0 to 35% A.S. fraction, as described in Figure 1,... 

Figure 3. Effect of ribosomal concentration on aminoacyl transfer to protein...

Aminoacyl transfer was found to be optimal with about 10 mg. of added pH 5 Supernatant protein. At much higher concentrations of this preparation, inhibition of amino acid incorporation was observed. When... 

Figure 5. Dependence of aminoacyl transfer on varying concentrations of transferases I and II...

Previous studies demonstrated that GTP was the only essential nucleotide in the aminoacyl transfer reaction (2, 10, 21, 22). The effect of varying concentrations of GTP on this reaction is presented in Figure 6. Maximum incorporation was obtained with as little as 0.05 jLimole of GTP per ml. in incubations with transferase I and soluble transferase n. When microsomal transferase II was used, the requirement for... 

The transfer of amino acids from sRNA to protein was dependent on the concentration of ribosomes, aminoacyl sRNA, and transferring enzymes. Aminoacyl transfer was observed with less than 0.05 pmole of GTP per ml., and the reaction exhibited a sulfhydryl requirement which was met by a variety of compounds. Transfer of several sRNA-bound C14-amino acids to ribosomal protein was observed with the puri-ified transferases I and n. 

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