Ribosome peptidyltransferase site

Some trichothecenes, a group of mycotoxins, have macrodiolide or macrotri-olide skeletons. Trichothecenes inhibited protein synthesis by binding to the ribosomal peptidyltransferase site [141]. Roritoxins (roritoxin A, 113) are 16-membered ring macrodiolides isolated from Myrothecium roridum [142]. Verru-carin A (114) is an 18-membered ring macrotriolide produced by Myrothecium spp. [143]. 

Mechanism of Action. THie earliest studies on the mechanism of action of lincomycin showed that lincomycin had the immediate effect on Staphjlococcus aureus of complete inhibition of protein synthesis (23). TThis inhibition results from the blocking of the peptidyltransferase site of the SOS subunit of the bacterial ribosome (24). Litde effect on DNA and RNA synthesis was observed. 

A careful stereochemical analysis has led to the conclusion that for all of the different aminoacyl groups to be able to react in the same way at the peptidyltransferase site and to all generate trans amide linkages, the torsion angles < ) and q/ of the resulting peptide must be approximately those of an a helix.388 Thus, the peptide emerging from the ribosome exit tunnel may be largely helical. 

Figure 29-16 Schematic diagram of the tmRNA structure and its function in the rescue of ribosomes stalled at the end of a messenger RNA that has been broken and has lost its inframe termination codon. After it binds into the ribosomal A site the tmRNA, which has been charged with alanine, undergoes the peptidyltransferase reaction and translocation to the P site. Then it lays down its mRNA-like coding sequence, which is used by the ribosome to add ten more amino acids to form the 11-residue C-terminal degradation signal A ANDENYALAA. This induces rapid degradation of the imperfect protein that has been formed.4363...

Deposition binding might cause a great conformational change in the overall three-dimensional structure essential for peptidyltransferase activity and the introduction of nascent peptide into the exit tunnel. Such modification also probably renders correct alignment of the peptidyl and aminoacyl substrates at the ribosome catalytic site, hindering peptide formation. In this connection, additional investigation is needed to obtain clear direct evidence. 

EF-Tu will bind to any aminoacylated tRNA other than tRNA the initiator tRNA (step c. Fig. 29-12), and carry it to the ribosome (step d), where it binds into the A site. There it is selected if it forms a proper base pair with the mRNA codon in the A site or is rejected if it does not. This decoding process involves both an initial step and a proofreading step. The aminoacyl-tRNA binds both to the decoding site in the 16S RNA and to the peptidyltransferase site in the 23S RNA. (See discussions on p. 1687.) The decoding site is on the platform at the upper end of helix 44 (Fig. 29-2). Nucleotide G1401 plays a crucial roie.375 vVhen one of the isoacceptor species of E. coU tRNA is irradiated with ultraviolet light, the... 

FIGURE 46-2 Inhibition of bacterial protein synthesis by chloramphenicol. Chloramphenicol binds to the 50S ribo-somal subunit at the peptidyltransferase site and inhibits the transpeptidation reaction. Chloramphenicol binds to the SOS ribosomal subunit near the site of action of clindamycin and the macrolide antibiotics. These agents interfere with the binding of chloramphenicol and thus may interfere with each other s actions if given concurrently. See Figure 46-1 and its legend for additional information. 

Translation ends when a termination codon enters the ribosomal A-site. Release of the newly synthesized protein chain from the ribosome is mediated by the action of the release factor RF-1 specific for the termination codons UAA and UAG or of the factor RF-2 specific for UAA and UGA. A third release factor and GTP stimulate this process. It is likely that the cleavage of tRNA from the completed protein chain is carried out by the same enzyme, namely the peptidyltransferase, which transfers the growing peptide chain from the P-site onto the aminoa< l-tRNA in the A-site. The termination process is completed by release of the newly synthesized protein chain, the de-ac lated tRNA and the messenger-RNA from the ribosome. Modification of the protein, e.g. acetylation and methylation, probably occurs when the growing chain is still on the ribosome and as soon as the amino acid to be modified become accessible for the appropriate non-ribosomal enzyme. 

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). 

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. 

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

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