Ribosomal sites aminoacyl

There are numerous examples of the inhibition of biosynthesis by fungicides. Some fungicidal secondary metabolites, like cyclohexi-mide and blasticidine, interfere with synthesis of peptide bonds at the ribosomal site (2). Another, kasugamycine, influences aminoacyl-t-RNA/ribosome interactions (3). Finally, another mechanism inhibiting protein biosynthesis is realized on the DNA/RNA- level by the... 

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).

During Chain Elongation Each Incoming Aminoacyl-tRNA Moves Through Three Ribosomal Sites... 

The translation step where proteins are assembled following the code of messenger RNA has a few instances of inhibition by alkaloids [361]. Emetine and tubulosine block peptide bond formation, acting similarly to the antibiotic cycloheximide by blocking translocation of the growing peptide chain from the A site to the P site of the ribosome. They evidently bind to a specific ribosomal site [362]. Homoharringtonine may act similarly [363]. Lycorine may act at the level of termination [364]. Narciclasine and related alkaloids of the Amaryllidaceae prevent binding of the 3 end of aminoacyl-tRNA to the peptidyl transferase site of the ribosome [365, 366]. Mescaline may act similarly [367]. 

The SOS subunit binds to the 30S initiation complex. It contains three sites for tRNA binding, called the P site (peptidyl), the A site (aminoacyl), and the E site (exit). When the two ribosomal subunits join, the AUG initiator codon with its bound tRNAf t aligns with the P site. 

Fig. 15.8. Initiation of protein synthesis. P site = peptidyl site on the ribosome A site = aminoacyl site on the ribosome (The A and P sites or portions of them are indicated by dashed lines) elF = eukaryotic initiation factor.

Peptidyl transferase center (PTC), a ribo-somal complex catalyzing peptide bond formation. The ribosomal PTC resides in the large ribosomal subunit and catalyzes both peptide bond formation and peptide release. The peptidyl transferase reaction involves aminolysis by the depro-tonated a-amino function of the A-site aminoacyl-tRNA of the ester bond linking the nascent peptide to the 3 hydroxyl of the 3 terminal ribose of the P-site tRNA. The formed short-lived tetrahedral reaction intermediate decomposes by donation a proton back to the leaving oxygen, yielding... 

A second site, called the A site (aminoacyl site), is located on the mRNA-ribosome complex next to the P site. The A site is where an incoming tRNA carrying the next amino acid will bond. Each of the tRNA molecules representing the 20 amino acids can fiy to fit the A site, but only the one with the correct anticodon that is complementary to the next codon on the mRNA will fit properly. 

The conformational change in the anticodon loop has been suggested to be an important event in protein biosynthesis (Woese, 1970 Lake, 1977). Thus in the Lake (1977) model, the anticodon conformation switches fi om a S stack to a 3 stack, after the correct recognition of the tRNA in the ribosome site. This would presumably allow the aminoacyl group to move toward the ribosome-bound peptidyl-tRNA. 

Our results suggest that initiator tRNA and chain-elon tor tRNAs can exist in both anticodon-loop conformations, although some of the elongator tRNAs (such as tRNA Fig. 6) do not appear to undergo a low-temperature conformational transition. Aminoacylated initiator tRNA goes only into the ribosomal A (aminoacyl) site and then during chain elongation later, translocates to the P site. 

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). 

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. 

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.

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. 

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