Puromycin, polypeptide chains

Comment A simple test that is often done is to treat the lysate with 30 fiM EDTA prior to loading on the gradient, which leads to the disassembly of polysomes due to chelation of Mg2+ (Fig. 6.4B). EDTA is, however, not a very specific reagent and instead more specific drugs such as puromycin, which causes polypeptide chain termination and disaggregates polysomes, may be used. 

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. 

Table 29.4). For example, the antibioticpuromycin inhibits protein synthesis by causing nascent prokaryotic polypeptide chains to be released before their synthesis is completed. Puromycin is an analog of the terminal aminoacyl-adenosine part of aminoacyl-tRNA (Figure 29.34). 

Figure 29.34. Antibiotic Action of Puromycin. Puromycin resembles the aminoacyl terminus of an aminoacyl-tRNA. Its amino group joins the carbonyl group of the growing polypeptide chain to form an adduct that dissociates from the ribosome. This adduct is stable because puromycin has an amide (shown in red) rather than an ester linkage.

Mechanism of action of puromycin. Puromycin is able to enter the ribosome A site and function as an aminoacyl tRNA analogue, resulting in polypeptide chain termination in both... 

Puromycin terminates the growing polypeptide chain by forming a peptide bond with its C-terminus, which prevents the formation of new peptide bonds (see Figure 12.14). 

Puromycin enters the A site, attaches to the polypeptide chain and then moves to the P site. The problem is it can neither accept the next... 

Studies with puromycin have provided further evidence for the existence of two binding sites on reticulocyte ribosomes. Apparently, puromycin can act as an acceptor and thus can form a peptide bond with an amino acyl tRNA in the donor position. Puromycin cannot, however, be bound enzymatically to ribosomes and as a result cannot act as a donor. Consequently, the elaboration of the polypeptide chain ends with puromycin. When the polypeptide is released from... 

It is not well known at what stage of the thyroglobu-lin molecule s biosynthesis tyrosine is iodinated. Three proposals have been submitted (1) iodination of the amino acid before its incorporation into the polypeptide chain (but puromycin does not impair iodination, and no enzymes capable of activating iodotyrosine have been found) (2) iodination of the finished tetra-meric globulin and (3) iodination of the 12 S subunits with concomitant condensation of the unit to yield new protein. However, there seems to be no doubt that thyroglobulin continues to be iodinated even after its excretion into the colloid, indeed the ratio... 

Puromycin has a structure closely analogous to the 3 terminus of a tyrosine-tRNA, and it inhibits protein synthesis by accepting, in place of an aminoacyl-tRNA, an incomplete polypeptide chain from ribosome-bound peptidyl-tRNA, thus prematurely terminating protein synthesis. We have found that irradiation at 2537 A (low-pressure Hg lamp) of solutions containing puromycin and ribosomes from E. coli leads to significant covalent incorporation of puromycin into the ribosome. For... 

Evidence that sugars were incorporated into nascent polypeptide chains was obtained by use of puromycin. Puromycin blocks protein synthesis by functioning as an analogue of amino acylated-tRNA and substitutes for the incoming amino acyl-tRNA as the acceptor... 

Puromycin inhibits protein synthesis in numerous cellular species (including eukaryotic cells). It was shown, by studies in subcellular systems for protein synthesis, that this antibiotic hinders the amino-acid transfer from the aminoacyl-transfer-RNA, to the growing polypeptide chains on the ribosomes. In 1959, Yarmolinsky and de la Haba postulated that puromycin acts as an aminoacyl-transfer-RNA analogue, because of its structural resemblance with the amino acid carrying acceptor extremity of an aminoacyl-transfer RNA (Fig. 1). This hypothesis is now completely confirmed. Puromycin induces the release of unfinished polypeptide chains, when acting in a protein-synthesizing system, and these polypeptides... 

Further,studying the effect of puromycin on growing polypeptide chains labelled with I C-leucine in the presence or absence of J-thiaproline,data were obtained indicating that j3-thiaproline once incorporated into polypep-... 

Fig. 16. Puromycin as naturally-occurring structural analog of the tRNA for phenylalanine and tyrosine. Incorporation into the growing polypeptide chain occurs through the encircled amino group Ri = polynucleotide chain of the tRNA R2 = H phenylalanine R2 = OH tyrosine.

An essential part of the rationale presented above under (a) consists of the identification of altered products of mitochondrial protein synthesis as a result of the mutation. Although this is not a sufficient criterion for mitochondrial specification (since an altered protein might arise as a result of a mutational alteration in a component of the mitochondrial protein-synthesizing machinery, i.e., one of the mt r- or tRNAs, (as in poky Neu-rospora), it is a necessary one. We have therefore devoted considerable effort to demonstrating the capability of the mitochondria in the mutant to perform some form of protein synthesis. We did this by showing that they were capable of incorporating (1) labeled formate into formylmethionyl-puromycin as a measure of mitochondrial polypeptide-chain initiations (see also next section) (2) labeled leucine into mitochondrial membrane proteins in a reaction that is insensitive to cycloheximide (CHX), but sensitive to chloramphenicol (CAP) and (3) that continued exposure of cells to the latter led to their conversion to petite phenocopies, s5 of characteristic aspects of their phenotype, such as the presence of cytochrome b, and that this change was reversed on removal of the inhibitor (see also Table I). 

This claim is based on the following observations (1) Like that of their prokaryotic ancestors, mitochondrial translation uses fMet-tRNA (rather than the Met-tRNA used by cytoribosomes) as the initiator. (2) It can do so successfully because the transformylase which converts the Met-tRNA into its fMet derivative is mitochondrial in its localization. (3) The formation of the initiation complex can be monitored by the transfer of labeled formate (f ) to f Met to puromycin, resulting in its quantitative conversion to f Met-puro —a reaction that is restricted both in vitro and in vivo to the mitochondrial fraction and can be shown to go on with linear kinetics for extended periods. (4) Retention of f Met on nascent polypeptide chains is restricted to mitochondrial polyribosomes and can be used as a specific means for the identification and characterization of the latter. (5) Mitochondria, at least of the yeast species examined by us, appear to be deficient in both a deformylase capable of removing formate from fMet, whether free or on polypeptides, as well as in peptidases capable of removing either this component itself or small peptides from the N-terminal end. (6) Initiation by fMet is absent in p" mutants. In principle then, presence of formate as N-terminal fMet in a polypeptide provides an unambiguous means for its identification as having been synthesized on mitoribosomes. In practice, although feasible, as will be shown in the next section, this is difficult because it requires the prior... 

We discussed earlier that one mechanism for the transfer of particular translation products across the membrane barriers is the process of vectorial translation as carried out by polysomes attached to the rough endoplasmic reticulum of secretory cells. An in vitro assay for this process has been devised by Redman and Sabatini. In order to carry out this assay, it is necessary to release labeled, nascent polypeptide chains from membrane-bound ribosomes by reaction with puromycin and assess their distribution between the medium and the membrane compartment. We followed this procedure to assay for vectorial release of nascent chains from bound 80 S polysomes attached to the outer mitochondrial membrane. 

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