Aminoacyl-tRNA, synthesis

Ibba, M. Soil, D. (2000) Aminoacyl-tRNA synthesis. Annu. 

Aminoacylation is a two-step process, catalyzed by a set of enzymes known as aminoacyl-tRNA synthetases. Twenty aminoacyl-tRNA synthetases reside in each cell, one per amino acid in the genetic code. In the first step of aminoacyl-tRNA synthesis, ATP and the appropriate amino acid form an aminoacyl adenylate intermediate. Inorganic pyrophosphate is released and subsequently broken down to free phosphate by the enzyme inorganic pyrophosphatase. The aminoacyl adenylate is a high-energy intermediate, and in the second step, the transfer of amino acids to the acceptor end of tRNA occurs without any further input of ATP, as shown in Figure 11-2. 

Unlike translational initiation, elongation, and aminoacyl tRNA synthesis, translational termination is a spontaneous process that does not require the input of energy (GTP hydrolysis). There are three codons on the mRNA that will trigger the end of translation when they appear at the A site on the ribosome UAA, UGA, and UAG. As the ribosome encounters these codons, one of two release factor proteins will bind at the A site and hy-... 

Feng L, Sheppard K, Namgoong S, Amhrogelly A, Polycarpo C, Randan L, Tumhula-Hansen D, Soil D. Aminoacyl-tRNA synthesis by pre-translational amino acid modification. RNA Biol. 2004 1 16-20. 

M. Ibba, A.W. Cumow, and D. Soil. 1997. Aminoacyl-tRNA synthesis Divergent routes to a common goal Trends Biochem. Sci. 22 39-42. (PubMed)... 

The sequential reactions that occur within the active site of aminoacyl-tRNA synthesis are (1) the formation of aminoacyl-AMP, which contains a high-energy mixed anhydride bond, and (2) linkage of the aminoacyl group to its specific tRNA. 

Ibba, M., and D. Soil. 2001.The renaissance of aminoacyl-tRNA synthesis. EMBO Rep. 2 382-387. 

H. Suga, Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme, /. Am. Chem. Soc. 2002, 124, 6834-6835. 

FIGURE 12.6 The aminoacyl-tRNA synthetase reaction, (a) The overall reaction. Everpresent pyrophosphatases in cells quickly hydrolyze the PPj produced in the aminoacyl-tRNA synthetase reaction, rendering aminoacyl-tRNA synthesis thermodynamically favorable and essentially irreversible, (b) The overall reaction commonly proceeds in two steps (i) formation of an aminoacyl-adenylate and (ii) transfer of the activated amino acid moiety of the mixed anhydride to either the 2 -OH (class I aminoacyl-tRNA synthetases) or 3 -OH (class II aminoacyl-tRNA synthetases) of the ribose on the terminal adenylic acid at the 3 -OH terminus common to all tRNAs. Those aminoacyl-tRNAs formed as 2 -OH esters undergo a transesterification that moves the aminoacyl group to the 3 -OH of tRNA Only the 3 -esters are substrates for protein synthesis. 

Puromycin. Puromycin (19), elaborated by S. alboniger (1—4), inhibits protein synthesis by replacing aminoacyl-tRNA at the A-site of peptidyltransferase (48,49). Photosensitive analogues of (19) have been used to label the A-site proteins of peptidyltransferase and tRNA (30). Compound (19), and its carbocycHc analogue have been used to study the accumulation of glycoprotein-derived free sialooligosaccharides, accumulation of mRNA, methylase activity, enzyme transport, rat embryo development, the acceptor site of human placental 80S ribosomes, and gene expression in mammalian cells (51—60). 

It has been known for some time that tetracyclines are accumulated by bacteria and prevent bacterial protein synthesis (Fig. 4). Furthermore, inhibition of protein synthesis is responsible for the bacteriostatic effect (85). Inhibition of protein synthesis results primarily from dismption of codon-anticodon interaction between tRNA and mRNA so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented (85). The precise mechanism is not understood. However, inhibition is likely to result from interaction of the tetracyclines with the 30S ribosomal subunit because these antibiotics are known to bind strongly to a single site on the 30S subunit (85). 

Transfer RNA (tRNA) serves as a carrier of amino acid residues for protein synthesis. Transfer RNA molecules also fold into a characteristic secondary structure (marginal figure). The amino acid is attached as an aminoacyl ester to the 3 -terminus of the tRNA. Aminoacyl-tRNAs are the substrates for protein biosynthesis. The tRNAs are the smallest RNAs (size range—23 to 30 kD) and contain 73 to 94 residues, a substantial number of which are methylated or otherwise unusually modified. Transfer RNA derives its name from its role as the carrier of amino acids during the process of protein synthesis (see Chapters 32 and 33). Each of the 20 amino acids of proteins has at least one unique tRNA species dedicated to chauffeuring its delivery to ribosomes for insertion into growing polypeptide chains, and some amino acids are served by several tRNAs. For example, five different tRNAs act in the transfer of leucine into... 

Tetracycline and its derivative doxycycline are antibiotics widely used in the treatment of bacterial infections. They also exert an antimalarial activity. Tetracyclines inhibit the binding of aminoacyl-tRNA to the ribosome during protein synthesis. 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid. 

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

Figure 38-8. Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labeled n - 1, n, n -I-1, etc, represent the amino acid residues of the newly formed protein molecule. EFIA and EF2 represent elongation factors 1 and 2, respectively. The peptidyl-tRNA and aminoacyl-tRNA sites on the ribosome are represented by P site and A site, respectively.

Figure 38-9. Diagrammatic representation of the termination process of protein synthesis. The peptidyl-tRNAand aminoacyl-tRNA sites are indicated as P site and A site, respectively. The termination (stop) codon is indicated by the three vertical bars. Releasing factor RF1 binds to the stop codon. Releasing factor RF3, with bound GTP, binds to RFl. Flydrolysisofthe peptidyl-tRNA complex is shown by the entry of HjO. N and C indicate the amino and carboxyl terminal amino acids, respectively, and illustrate the polarity of protein synthesis.

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

Of the fonr possible optical isomers of chloramphenicol, only the o-threo form is active. This antibiotic selectively inhibits protein synthesis in bacterial ribosomes by binding to the 50S subunit in the region of the A site involving the 23 S rRNA. The normal binding of the aminoacyl-tRNA in the A site is affected by chloramphenicol in such a... 

YAMAGUCHI M and suGiMOTO E (2000) Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells Activation of aminoacyl-tRNA synthetase. Mol Cell Biochem 214, 97-102. 

If a translation reaction directed by the universal 027IF2Cp(A) mRNA is carried out in the presence of four precharged aminoacyl-tRNAs (fMet-tRNA, Phe-tRNA, Thr-tRNA, and Ile-tRNA) in amounts sufficient to ensure the synthesis of the 027 peptide (which contains only these amino acids) even in the presence of an aminoacyl-tRNA inhibitor, the system will be able to detect an aminoacylation inhibitor in a library of natural or synthetic products through the selective inhibition of IF2C domain synthesis. Thus, if the synthesis of the 027 and IF2C peptides is measured in parallel, a general inhibitor of translation would be expected to inhibit the synthesis of both products, while an aminoacylation inhibitor would inhibit... 

Although aminoacyl-tRNA synthetases are necessary for protein synthesis in all tissues, their importance in chemical carcinogenesis is difficult to assess. Mutation induction by this pathway has been studied extensively (123), yet metabolic activation in a carcinogen-target tissue has not been demonstrated. The only exception is hepatic prolyl-tRNA synthetase activation of N-hydroxy-Trp-P-2 however, hepatic O-acetylation of this substrate also occurs to an appreciable extent (12). Further investigations involving the use of specific enzyme inhibitors would be helpful in addressing this problem. 

Activation of individual amino acids occurs in the synthesis of aminoacyl tRNA. This process bums two ATP equivalents (forms pyrophosphate and AMP) and connects a specific amino acid to a specific tRNA. 

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