Puromycin reaction

The peptidyltransferase activity of ribosomes can be segregated from other translation reactions by the so-called puromycin reaction [129] which monitors the formation of [acetyl-aminoacyl]-puromycin or [peptidyl]-puromycin from puromycin and either [acetyl-aminoacyl]-tRNA or [peptidyl]-tRNA. In its simplest form (termed uncoupled or 30S-subunit-independent peptidyltransferase) the reaction requires large ribosomal subunits and an organic solvent (ethanol or methanol) which is presumably needed to promote the binding of tRNA and puromycin to the 50S subunit. In the absence of organic solvents, however, the reaction (then termed coupled or 30S-subunit-dependent... 

It is usually accepted that the 16-membered-ring macrolides inhibit peptidyltransferase activity [79, 88, 89], because they inhibit puromycin reaction although poorly. The reaction is used as an assay system for peptidyltransferase activity, because puromycin characteristically interrupts peptide bond formation by virtue of its structural similarity to the 3 end of aminoacyl-tRNA. Puromycin enters the A site (the so-called aminoacyl site) on the ribosome and is incorporated into either a nascent polypeptide or into A-acylaminoacylate, consequently causing premature release of puromycinyl polypeptide or A-acylaminoacyl-puromycin from the ribosome. 

On the other hand, the inhibitory effect of erythromycin, a 14-membered-ring macrolide, on such a peptidyltransferase reaction is markedly diminished in terms of the character of a substrate. Erythromycin inhibits poly(A)-dependent polymerization of a transferred substrate such as lysine residue linked to tRNA but not other oligonucleotide-dependent polymerization of an amino acid linked either to tRNA or to oligonucleotides such as CACCA and UACCA. It has been shown that the transfer of A-acylaminoacyl residues to puromycin (puromycin reaction) is usually stimulated by erythromycin [88, 89, 95]. Igarashi et al. [96] have also confirmed these findings. That is to say, they found that erythromycin inhibits the release of a deacylated tRNA from the P site of ribosome. The release of such a deacylated tRNA from the P site and the translocation of peptidyl-tRNA from the A site to the P site of ribosome occurs concomitantly when EF-G catalyzes the GTP-dependent movement of the ribosome and the codon-anticodon-linked mRNA-peptidyl-tRNA complex. 

Moreover, eRF enhances eIF-2 activity to that of the complex, suggesting complex formation between eIF-2 and eRF as an intermediary step in the initiation pathway. Therefore, conditions for complex formation between eIF-2 and eRF have been investigated and the effects of HRI-mediated phosphorylation of eIF-2 have been included. The results are shown in Fig. 10, from which we can conclude the following the preparations used in this experiment are essentially free of the eIF-2 eRF complex (Fig. lOA). Thus the complex found upon incubation of C-labeled eIF-2 with eRF is newly formed (Fig. lOB). In addition no complex is formed with phosphorylated eIF-2 which is in agreement with the fact that eRF failed to stimulate the activity of eIF-2a-P in the methionyl-puromycin reaction (see Fig. 9). Moreover the eIF-2 moiety in the complex can be replaced by free eIF-2 (Fig. IOC). Furthermore we checked whether the a suhunit of eIF-2 is more... 

As indicated above, it is possible that codons bind to ribosomes at two positions corresponding to donor and acceptor sites. The nature of the site occupied by attached oligonucleotides has only been tentatively determined by testing the reactivity in the puromycin reaction of the aminoacyl-tRNA bound to the modified ribosomes. In another case, the ability of ApUpGpU -modified 70 S ribosomes to bind Met-tRNA in the presence of EF-Tu has been considered indicative of acceptor site location of the bound codon. ... 

Glutamine and ATP analogues were useful to probe the reaction mechanism, but these inhibitors are likely to interfere with many other enzymes acting on the same substrates. More recently, analogues of puromycin (22) (Table 5) were synthesized and evaluated as mechanism-based selective inhibitors of H. pylori... 

Figure 29-13 (A) Structure of expected intermediate with tetrahedral C-atom in peptidyltransferase reaction with a tRNA, with a minihelix analog, or with the antibiotic puromycin. (B) Transition-state (or bisubstrate) analog formed with puromycin and a mimic of the CCA end of a tRNA. See Box 29-B.

Baker s original work concerned the synthesis of 3-amino-3-deoxy-D-ribose, a component residue of the antibiotic puromycin.29 Two independent syntheses were achieved, starting from L-arabinose and D-xylose, respectively. The main steps in the reaction schemes are outlined in the following reaction sequences. 

In a dramatic demonstration of the use of a scavenger Nicolson et al. (1982) demonstrated that a long-lived intermediate formed when p-azi-do[3H]puromycin was used to label F.. coli ribosomes was diverted from its reaction with polypeptide SI8 by 2 mM P-mercaptoethanol. SI8 was the... 

It competes with the latter as an acceptor in the peptidyl transfer reaction. The growing chain is transferred to the NH2 group of puromycin and is prematurely terminated. 

The three antibiotic inhibitors of translation that will be used in this experiment are chloramphenicol, cycloheximide, and puromycin (Fig. 23-10). Chloramphenicol is specific for prokaryotic ribosomes, blocking the transfer of the peptide on the tRNA at the P site to the amino acid linked to the tRNA at the A site (the peptidyl transfer reaction). Since the source of the ribosomes used in this experiment is wheat germ (eukaryotic), we would predict that chloramphenicol would not have a great effect on translation. The mechanism of cycloheximide-mediated inhibition is the same as that described above for chloramphenicol, except for the fact that it is specific for the 80S eukaryotic ribosome. Puromycin is a more broad translational inhibitor, effective on both eukaryotic and prokaryotic ribosomes. It acts as a substrate analog of aminoacyl tRNA. When it binds at the A site of the ribosome, it induces premature termination of translation (Fig. 

Compare the relative level of 3H-poly-Phe present in vial 11 compared to that in vial 16. Explain your results in terms of the components that were present in these two reactions, and your knowledge of the mechanism of action of puromycin. 

The reactions of phase 2 relate to the attachment of the bridge-carbohydrate residues to the polypeptide chain. There is evidence showing that this addition occurs while the polypeptide chain is still attached to, or perhaps still being synthesized on, the ribosomes.101-103 Thus, 14C-labeled 2-amino-2-deoxy-D-glucose, injected into the circulatory system of the rat, was incorporated into protein in the ribosomes of the rough endoplasmic-reticulum of the liver. Administration of puromycin caused release of the 14C-labeled glycoprotein, which could be isolated by acid-precipitation methods. Examination of the radioactivity data revealed that the subcellular structures most actively involved in glycoprotein synthesis were the ribosomes bound to the membrane, and not free polysomes. 

The use of primary and secondary amines usually results in the displacement reaction without difficulty. Thus, the base component 2 of puromycin is formed from 6-chloropurine with methanolic dimethylamine at 100 C. ... 

Earlier antitrypanosomiasis agents included the aminonaphthalenes trypan blue and Afridol violet. They were followed by the amide suramin79 (see Chapter 1). Heterocyclic aromatic amines include quinapyramine (138) and puromycin (139). Starting with 2-aminobiphenyl (140), a five-step reaction yields the phenanthridine dimidium (141) (Scheme 28). 

The 2-oxabicyclo[3.1.0]hexane nucleoside 166 was obtained via a Simmons-Smith type cyclopropanation reaction of intermediate 164, followed by glycosidation with several natural heterocyclic bases <05JOC6891 05NNNA383>. A restricted version of puromycin built on a bicyclo[3.1.0]hexane template was synthesized by Choi via Mitsunobu coupling of a 3-azido-substituted carbocyclic moiety with 6-chloropurine to give compound 167 <02OL589>. 

A good example of translational inhibition is the mechanism by which puromycin mimics the structure of the aminoacyl group of aminoacyl-tRNA, as shown in Figure 26.15. Puromycin is an aminoacyl-tRNA analogue that is able to bind to the A site of both prokaryotic and eukaryotic ribosomes, even without a corresponding tRNA, and thus act as an acceptor for the peptidyl transferase reaction. 

---