4 Important Inhibitors of Protein Synthesis

Many substances are known to act as inhibitors of var­ious stages of protein synthesis. Included among these are a number of antibiotics produced by one strain of microorganism and lethal to other strains of the same or a different species.

Some of the best understood in­hibitors of protein synthesis are listed in Table 22-10. Because the actions of many of these inhibitors are quite specific, they have proved to be extremely useful tools in the step-by-step elucidation of the mechanism of protein synthesis.

1. Inhibitors of both Prokaryotic and Eukaryotic Protein Synthesis:

Aurintricarboocylic acid inhibits formation of the initi­ation complex by preventing the association of mRNA with the small ribosomal subunit. Inhibitors of initia­tion are readily distinguished from inhibitor’s blocking other stages of protein synthesis because of the delay effect that follows their administration, that is, protein synthesis continues for a short time after administra­tion of the inhibitor, because peptide chains whose growth had already begun are unaffected and grow to completion.

Edeine, a polypeptide isolated from Bacil­lus brevis, inhibits the binding of aminoacyl-tRNA and N-formylmet-tRNA^M_f^et (in prokaryotes) to the small subunit. Fusidic acid is a steroidal antibiotic; in pro­karyotes, it inhibits the binding of aminoacyl-tRNA to the ribosome, whereas in eukaryotes, it inhibits trans­location by reacting with elongation factor.

Puromycin was one of the first inhibitors of protein synthesis to have its specific effect determined. This antibiotic mimics aminoacyl-tRNA and binds to the free A site of ribosomes engaged in protein synthesis. Catalytic formation of a bond between the nascent polypeptide and puromycin is followed by the release of the peptidyl-puromycin from the ribosome, as no further elongation is possible.

The specific effects of puromycin have been used to advantage for studies of nascent chain length, the kinetics of chain elongation, and the identification of the effects of other antibiot­ics. Tetracycline inhibits protein synthesis by blocking aminoacyl-tRNA binding to the small subunit.

2. Inhibitors Specific for Prokaryotes:

Chloramphenicol (Chloromycetin) binds to the large subunit of prokaryotic ribosomes and interferes with the functioning of peptide synthetase, thereby inhibit­ing chain elongation. Colicin E3 inhibits protein syn­thesis in prokaryotes by interfering in some manner with the functioning of the small subunit. Erythromy­cin binds to ribosomes that are not engaged in protein synthesis, preventing their potential participation, but does not bind to ribosomes containing nascent chains (i.e., ribosomes that are part of a functioning polysome). Streptomycin was one of the earliest dis­covered antibiotics and was employed as an agent against bacterial infection for many years before its specific chemical actions were known.

Streptomycin binds to protein S12 of the small ribosome subunit, causing release of N-formylmet-tRNA^M_f^et from initia­tion complexes (thereby preventing initiation of chain growth) and also causing misreading of the codons of mRNA by ribosomes already involved in chain elonga­tion.

3. Inhibitors Specific for Eukaryotes:

Anisomycin is an antibiotic produced by Streptomyces that inhibits peptide bond formation when bound to the small ribosomal subunit. Cycloheximide binds to the large subunit, preventing the translocation of tRNA in the A site to the P site. Diphtheria toxin (produced by a strain of Corynebacterium diphtherial) inhibits protein synthesis through its action on EF-2 (translocase). EF-2 exists in cells in two forms— ribosome-bound and free. Diphtheria toxin acts enzy­matically to alter free EF-2, rendering the factor in­active. Ribosome-bound EF-2 is not susceptible to inactivation by the toxin.

Pactamydn (produced by a strain of Streptomyces) binds to free small subunits (not to small subunits al­ready part of polysomes), where it prevents initiation by inhibiting binding of met-tRNA j and formation of the initiation complex. The toxic effects of ricin (a protein present in the castor bean) have been known for nearly a century.

Ricin consists of two polypeptide chains (linked by disulfide bridges), one of which acts as the inhibitor once incorporated into the cell. Ricin acts on the large subunit, preventing formation of the 80 S initiation complex. Like ricin, sodium fluoride acts as an inhibitor or initiation; NaF blocks addition of the large subunit to mRNA.

Sparsomycin, another antibiotic produced by Strep- tomyces, inhibits the association of the amino acid moi­ety of aminoacyl-tRNA from binding to the large sub- unit and, in so doing, blocks peptide synthetase. THchodermin is the only chemical compound so far identified as a specific inhibitor of the termination stage of polypeptide synthesis.

4. Inhibitors of Organeiiar Protein Synthesis:

Protein synthesis by mitochondrial and chloroplast ribosomes is also subject to inhibition by certain antibi­otics and other chemicals. Shortly after the initial demonstration of organellar protein synthesis, it was found that chloramphenicol, a strong inhibitor of prokaryote protein synthesis, blocks synthesis in mito­chondria and chloroplasts, whereas cycloheximide, which blocks eukaryote cytoplasmic ribosomal protein synthesis, is without effect on mitochondrial and chlo­roplast synthesis.

These observations provided added credence for the notion that prokaryotic cells, mito­chondria, and chloroplasts have a common evolution­ary origin. It is now clear, however, that the picture is considerably more complex. For example, streptomy­cin, which inhibits prokaryotic protein synthesis, fails to inhibit mitochondrial protein synthesis in yeast cells.

Other antibiotics inhibit mitochondrial protein synthesis but have no effect on prokaryotes. Erythro­mycin inhibits the synthesis of proteins in prokary­otes, yeast mitochondria, and chloroplasts but fails to block protein synthesis in mammaliam mitochondria.

The latter observation suggests that the nature of mitochondrial protein synthesis varies among differ­ent groups of eukaryotes. In general, mitochondria from higher eukaryotes are more resistant to inhibi­tors of prokaryotic protein synthesis than are mito­chondria from lower eukaryotes. In chloroplasts, pro­tein synthesis is inhibited by the same agents that block this process in prokaryotic cells.

The differential sensitivity of eukaryotic cytoplas­mic and mitochondrial ribosomes to specific inhibitors provides a means for examining the sources of certain mitochondrial proteins. The synthesis of a mitochon­drial protein in the presence of cycloheximide (a cyto­plasmic inhibitor) indicates that the mitochondria are the source of the protein, whereas synthesis of the protein in the presence of chloramphenicol indicates that the mitochondrial protein is produced in the cyto­plasm and then moves to the mitochondria.

Experi­mentally, determinations of this sort are carried out by incubating cells in media containing both radioactively labeled amino acids and inhibitor. The synthesis of the mitochondrial protein is manifested by the ap­pearance of radioactivity in the protein later isolated from the mitochondria.

Using this approach, it has been possible to show that perhaps 85 to 90% of all mitochondrial ribosomal proteins are synthesized in the cytoplasm and then are imported into the mitochondria, where, together with mitochondrial rRNA, they are assembled into ribosomes. Of the seven polypeptides that make up the enzyme cytochrome oxidase, four are synthesized in the cytoplasm and three are synthesized in the mi­tochondria.