Protein synthesis peptidyl-tRNA

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 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. 505 ribosomal subunit and prevents attachment of aminoacybtransfer RNA (tRNA) to its binding site. The amino acid substrate is unavailable for peptidyl transferase and peptide bond formation. 

When any of the three stop (termination or nonsense) codons moves into the A site, peptidyl transferase (with the help of release fector) hydrolyzes the completed protein from the final tRNA in the P site. The mRNA, ribosome, tRNA, and factors can aU be reused for additional protein synthesis,... 

The final step in protein biosynthesis is chain termination. Natural mRNA molecules contain termination codons UAA, UGA, or UAG There are no tRNAs that have anticodons which are complementary to these codons. When the growing peptide chain encounters one of these termination codons, the peptidyl-tRNAis transferred to water instead of another aminoacyl-tRNA. The peptidyl-tRNA is hydrolyzed to free the completed protein and the tRNA. Chain termination completes protein synthesis. 

Figure 12-2. Formation of the initiation complex for protein synthesis. Several eukaryotic initiation factors (elFs) ensure proper assembly at each step. The initiator Met-tRNA is bound in the peptidyl site of the SOS complex with its anticodon (black stripes) base paired to the AUG start codon (gray box) of the mRNA.

Dealing with lost peptidyl-tRNAs and broken transcripts. Many problems arise during protein synthesis. For example, a peptidyl-tRNA may become detached from a ribosome. In E. coli this seems to happen most frequently with peptidyl-tRNA y5. A 193-residue peptidyl-tRNA hydrolase is essential for life 434-436 It releases the tRNA for reuse, recycling all peptidyl-tRNAs other than formylmethionyl-tRNA. Perhaps the enzyme is essential because detached peptidyl-tRNAs are toxic, but it is more likely to be to avoid a shortage of free tRNAl vs.434... 

Elongation factors. Protein factors uniquely required during the elongation phase of protein synthesis. Elongation factor G (EF-G) brings about the movement of the peptidyl-tRNA from the A site to the P site of the ribosome. 

Steps in bacterial protein synthesis and targets of (1) chloramphenicol (2) macrolides, clindamycin, and type B streptogramins and (3) tetracyclines. The 70S ribosomal mRNA complex is shown with its 50S and 30S subunits. The peptidyl tRNA at the donor site donates the growing peptide chain to the aminoacyl tRNA at the acceptor site in a reaction catalyzed by peptidyl transferase. The tRNA, discharged of its peptide, is released from the donor site to make way for translocation of the newly formed peptidyl tRNA. The acceptor site is then free to be occupied by the next "charged" aminoacyl tRNA. 

There are a number of sites within the sequence of protein synthesis where antibiotics can act. These include (1) inhibition of the attachment of mRNA to 30S ribosomes by aminoglycosides (2) inhibition of tRNA binding to 30S ribosomes by tetracyclines (3) inhibition of the attachment of mRNA to the 50S ribosome by chloramphenicol and (4) erythromycin inhibition of the translocation step by binding to 50S ribosomes, thus preventing newly synthesized peptidyl tRNA moving from the acceptor to the donor site. 

Figure 29.24. Mechanism of Protein Synthesis. The cycle begins with peptidyl-tRNA in the P site. An aminoacyl-tRNA binds in the A site. With both sites occupied, a new peptide bond is formed. The tRNAs and the mRNA are translocated through the action of elongation factor G, which moves the deacylated tRNA to the E site. Once there, it is free to dissociate to complete the cycle.

After the correct aminoacyl-tRNA has been placed in the A site, the transfer of the polypeptide chain from the tRNA in the P site is a spontaneous process, driven by the formation of the stronger peptide bond in place of the ester linkage. However, protein synthesis cannot continue without the translocation of the mRNA and the tRNAs within the ribosome. The mRNA must move by a distance of three nucleotides as the deacylated tRNA moves out of the P site into the E site on the 30S subunit and the peptidyl-tRNA moves out of the A site into the P site on the 30S subunit. The result is that the next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA. 

C. mRNA supplies the codons, aminoacyl-tRNA and GTP provide energy, peptidyl transferase catalyzes the formation of peptide bonds, and elongation factor 2 translocates the peptidyl-tRNA. Formylmethionyl-tRNA is involved in initiation of protein synthesis in prokaryotes. 

D. Protein synthesis is normally terminated by release factors, not by nuclease cleavage of mRNA or peptidyl-tRNA that cannot bind at the P site. UGA and UAG are stop codons, but AUG is the initiation codon. 

Puromycin is a specific metabolic inhibitor of protein synthesis and acts as an aminoacyl-tRNA analog and peptidyl acceptor. The latter causes premature chain... 

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