Peptidyl-tRNA, dissociation from ribosomes

Karimi R, Ehrenberg M (1994) Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes. Eur J Biochem 226 355-360 Karimi R, Ehrenberg M (1996) Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes. EMBOJ 15 1149-1154... 

Puromycin, made by the mold Streptomyces al-boniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the 3 end of an aminoacyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 27-31). However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. 

V. Heurgue-Hamard, R. Karimi, L. Mora, J. MacDougall, C. Leboeuf, G. Grentzmann, M. Ehrenberg, and R.H. Buckingham. 1998. Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome EMBO J. 17 808-816. (PubMed)... 

When a stop codon appears at the A site translation is terminated. There are no tRNA s that recognize stop codons. Instead releasing factors, eRF, recogiiize the stop codon. The releasing factors along with peptidyl transferases and GTP catalyze the hydrolysis of the bond between the polypeptide chain and the tRNA. The protein and tRNA disassociate from the P site and the ribosome dissociates into the 40S and 60S subunits releasing the mRNA. 

Menninger and Otto [101] proposed a major inhibitory mechanism common to probably all macrolide antibiotics. In E. coli mutants with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase EC 3.1.1.29), they observed that peptidyl-tRNA accumulates at a nonpermissive temperature (40°C) and that the cells die. The accumulation at a high temperature was enhanced when the cells were pretreated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30°C). Based on their observations, they suggested that stimulated dissociation of peptidyl-tRNA from ribosomes is the major mechanism of action of macrolide antibiotics. Their observations agree with recent results showing that a macrolide antibiotic binds to peptidyltransferase in ribosome. 

More detailed study of the stimulated dissociation of peptidyl-tRNA from ribosomes and of the bactericidal effect of the semisynthetic antibiotics on susceptible bacteria is needed to determine whether there is a relationship between... 

In prokaryotes, two release factors have been identified, one (RFl) recognizing UAA and UAG, the other (RF2) functioning with UGA. Ribosomal binding and release of RFl and RF2 are stimulated by a third factor, RF3, which interacts with GTP and GDP. In eukaryotic cells such as reticulocytes, one release factor (eRF) has been found to function with all three termination codons, and the binding of this factor to ribosomes is stimulated by GTP but not GDP. Although the details are not entirely clear, GTP hydrolysis appears to be required for the release of the finished polypeptide chain by cleavage of the peptidyl-tRNA bond and completion of the termination process leading to dissociation of the release factor from the ribosome. 

Caskey (1977) has proposed the following model for termination. With addition of the final amino acid to the nascent polypeptide, the chain moves from the A site to the P or D site, with the resultant placement of a terminator codon in the A site. Since this codon does not specify an amino acid, RF and GTP bind to the A site. It is proposed that RF activates the peptidyl transferase activity on the 60 S subunit which mediates hydrolysis of the ester bond of peptidyl-tRNA. In this model, GTP hydrolysis permits dissociation of the RF. This process has been considered in greater detail by Mazumder and Szer (1977), as well as by Caskey (1977). The role of GTP in this process appears to be analogous to that in initiation and elongation it is not required for binding of soluble factors to the ribosome but rather is utilized to mediate dissociation of these factors. 

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