Translocation, peptidyl-tRNA

Macrolides, lincosamides and streptogramins are protein biosynthesis inhibitors that bind to 50S subunit of the ribosome and inhibit peptidyl tRNA translocation from the A-site to the P-site." Macrolides have a glycosylated 14-, 15- or 16-membered lactone ring structure and are produced by several species of Streptomyces. Lincosamide antibiotics were isolated initially from Streptomyces lincolnensis but later isolated from different species of Streptomcyces. Streptogramins were also isolated from Streptomycesgraminofaciens and subsequently from several different Streptomyces species. There are two structurally different streptogramins, A and B they are bacteriostatic individually and can be bactericidal when combined. 

For processive peptide polymerization, the rihosome has to move along the mRNA. Following peptide bond formation, the rihosomal A site is occupied hy a peptidyl-tRNA whereas the P site contains a deacylated tRNA. During translocation, the complex of the two tRNAs with the mRNA has to move relative to the ribosome to... 

In the translocation step, the ribosome moves exactly three nudeotides (one codon) along the message. This moves the growing peptidyl-tRNA into the P site and aligns the next codon to be translated with the empty A site. 

A nasopharyngeal swab obtained from a 4-month-old infrint with rhinitis and paroxysmal coughing tested positive upon culture for Bordetella pertussis. He was admitted to the hospital for therapy with an antibiotic that inhibits the translocation of peptidyl-tRNA on 70S ribosomes, This patient was most likely treated with... 

Then the peptidyl-tRNA at the A site is translocated to the P site by the ribosome moving along the mRNA a codon at a time, exposing the A site for a new aminoacyl-tRNA appropriate for the particular codon, and a repeat of the elongation process occurs. The cycles of elongation and translocation continue until a termination codon is reached, and the peptide or protein is then hydrolysed and released... 

The ribosome then translocates to the next codon, with the peptidyl-tRNA shifting from the A site to the P site and the now uncharged tRNA exiting the ribosome from the E site. 

Elongation factor EF-G and translocation. The third step in the elongation sequence on ribosomes (Fig. 29-12, step g) depends upon EF-G, a monomeric GTP-binding protein with a sequence homologous with that of other members of the G protein family. It apparently utilizes the Gibbs energy of hydrolysis of GTP to GDP to drive translocation of the peptidyl-tRNA from the A site to the P site (Fig. 29-12) and of the previously utilized (de-acylated) tRNA to the exit site. 

The translocation reaction in E. coli. The translocation reaction occurs immediately after peptide synthesis. It involves displacement of the discharged tRNA from the P site and concerted movement of the peptidyl-tRNA and mRNA so that the peptidyl-tRNA is bound to the P site and the same three nucleotides in the mRNA. The A site is vacated and ready for the addition of another aminoacyl-tRNA. Translocation in eukaryotes is similar except that the EF-2 factor is involved instead of the EF-G factor. 

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. 

After translocation, the A site is empty and ready to receive the next aminoacyl-tRNA. The A site and the E site cannot be occupied simultaneously. Thus the deacylated tRNA is released from the E site before the next aminoacyl-tRNA binds to the A site to start a new round of elongation. Elongation continues, adding one amino acid to the C-terminal end of the growing polypeptide for each codon that is read, with the peptidyl-tRNA moving back and forth from the P site to the A site as it grows. 

Translocation, however, requires the input of energy (again, in the form of GTP) with the participation of the elongation factor EF-G. The translocation reaction moves the peptidyl-tRNA from the A-site to the P-site. The uncharged tRNA is removed from the P-site (it remains bound at an Exit or E-site for another cycle of elongation), while the ribosome and mRNA move relative to each other. This is shown in Figure 11-8. 

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. 

In translocation, the peptidyl-tRNA is shifted from the A site to the P site on the ribosome. What happens to the peptidyl-tRNA anticodon-codon interaction ... 

In the first step of the peptidyl transferase reaction, a peptidyl tRNA molecule is bound in the P-site with its nascent peptide extending down the peptide exit tunnel (Fig. 4.1). An elongation factor binds to a factor binding site (FBS) and positions an aminoacyl-tRNA in the A-site. The a amino group of the aminoacyl-tRNA nucleophilically attacks the ester bond which connects the peptide to the tRNA bound in the P-site (Fig. 4.2). The ester bond is broken as an amide bond forms, and the peptide becomes one amino acid longer, and is now attached to the tRNA that in the A-site. Translocation of the products follows peptide bond formation, as the newly formed deacylated- tRNA of the P-site moves into the E-site, and as the newly elongated peptidyl-tRNA moves from the A-site into the P-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. 

The ribosome includes three sites for tRNA binding called the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site. With a tRNA attached to the growing peptide chain in the P site, an aminoacyl-tRNA binds to the A site. A peptide bond is formed when the amino group of the aminoacyl-tRNA nucleophically attacks the ester carbonyl group of the peptidyl-tRNA. On peptide-bond formation, the tRNAs and mRNA must be translocated for the next cycle to begin. The deacylated tRNA moves to the E site and then leaves the ribosome, and the peptidyl-tRNA moves from the A site into the P site. 

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