Aminoacyl-tRNA 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.

Murray, J. B. Meroueh, S. O. Russell, R. J. Lentzen, G. Haddad, J. Mobashery, S. Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site. Chem. Biol. 2006, 13, 129-138. 

BINDING OF ANTIBIOTICS TO THE AMINOACYL-TRNA SITE OE BACTERIAL RIBOSOME... 

Codon-anticodon recognition between mRNA and the stem loop of tRNA on the ribosomal snrface brings one amino acid at a time for the formation of amide bonds at the top of ribosomal helix 44, a site known as the aminoacyl-tRNA site (A-site). It is not surprising that the A-site is also the binding site for... 

Fig. 2. Schematic of a prokaryotic 70S ribosome showing the peptidyl-tRNA site (P site), aminoacyl-tRNA site (A site) and exit site (E site).

B. Llano-Sotelo, E.F. Azucena Jr, L.P. Kotra, S. Mobashery, C.S. Chow, Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site, Chem. Biol. 2002, 9, 455-463. 

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). 

Aminohexose Nucleosides. The 4-aminohexose nucleosides (128—140) are Hsted in Table 7 (1—4,240—242). A biosynthetic relationship between the 4-aminohexose peptidyl nucleoside antibiotics and the pentopyranines has been proposed (1). The 4-aminohexose pyrimidine nucleoside antibiotics block peptidyl transferase activity and inhibit transfer of amino acids from aminoacyl-tRNA to polypeptides. Hikizimycin, gougerotin, amicetin, and blasticidin S bind to the peptidyl transferase center at overlapping sites (243). 

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). 

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. 

Elongation is a cycUc process on the ribosome in which one amino acid at a time is added to the nascent peptide chain. The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation. 

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. 

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... 

After formation of the initiation dipeptide, the first EF-G-dependent translocation allows binding of the third aminoacyl-tRNA in the A-site so that a tripeptide is formed. The apparent rate of this event may depend upon the nature of the initiation complex initially formed, being slower, for instance, with those containing mRNAs with an extended SD sequence than with those having either very short or no SD complementarity (C. O. G. and M. Rodnina, unpublished results). Furthermore, very powerful translocation inhibitors may block tripeptide formation to such an extent that they mimic translation initiation inhibitors. 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

Aside from these relatively direct applications of site-directed mutagenesis, combination of recombinant DNA techniques with other experimental strategies will no doubt prove to be of increasing importance. If the gene of interest can be expressed with sufficient efficiency in auxotrophs, then proteins in which selected amino acids are isotopically enriched [12] may be produced to increase the sensitivity and selectivity of magnetic resonance techniques. Alternatively, amino acid analogues that are recognized as substrates by aminoacyl tRNA synthetases may be incorporated randomly in place of the true substrate amino... 

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