Many chemicals that inhibit various aspects of protein synthesis have been identified. These chemicals are powerful experimental tools and clinically useful drugs.

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The differences between eukaryotic and bacterial ribosomes can be exploited for the development of antibiotics (Table 30.4). For example, the antibiotic streptomycin, a highly basic trisaccharide, interferes with the binding of fMet-tRNA to ribosomes in bacteria and thereby prevents the correct initiation of protein synthesis. Other aminoglycoside antibiotics such as neomycin, kanamycin, and gentamycin interfere with the interaction between tRNA and the 16S rRNA of the 30S subunit of bacterial ribosomes. Chloramphenicol acts by inhibiting peptidyl transferase activity. Erythromycin binds to the 50S subunit and blocks translocation.

The antibiotic puromycin inhibits protein synthesis in both bacteria and eukaryotes by causing nascent polypeptide chains to be released before their synthesis is completed. Puromycin is an analog of the terminal part of aminoacyl-tRNA (Figure 30.30). It binds to the A site on the ribosome and blocks the entry of aminoacyl-tRNA. Furthermore, puromycin contains an α-amino group. This amino group, like the one on aminoacyl-tRNA, forms a peptide bond with the carboxyl group of the growing peptide chain. The product, a peptide having a covalently attached puromycin residue at its carboxyl end, dissociates from the ribosome. No longer used medicinally, puromycin remains an experimental tool for the investigation of protein synthesis. Cycloheximide, another antibiotic, blocks translocation in eukaryotic ribosomes, making a useful laboratory tool for blocking protein synthesis in eukaryotic cells.

Many antibiotics, harvested from bacteria for medicinal purposes, are inhibitors of bacterial protein synthesis. However, some bacteria produce protein-synthesis inhibitors that inhibit eukaryotic protein synthesis, leading to diseases such as diphtheria, which was a major cause of death in childhood before the advent of effective immunization. Symptoms include painful sore throat, hoarseness, fever, and difficulty breathing. The lethal effects of this disease are due mainly to a protein toxin produced by a phage infecting Corynebacterium diphtheriae, a bacterium that grows in the upper respiratory tract of an infected person. A few micrograms of diphtheria toxin is usually lethal in an unimmunized person because it inhibits protein synthesis. The toxin consists of a single polypeptide chain that is cleaved into a 21-kDa A fragment and a 40-kDa B fragment shortly after entering the cell. The role of the B-fragment in the intact protein is to bind to the cell, enabling the toxin to enter the cytoplasm of its target cell. The A fragment catalyzes the covalent modification of EF2, the elongation factor catalyzing translocation in eukaryotic protein synthesis, resulting in protein synthesis inhibition and cell death.

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A single A fragment of the toxin in the cytoplasm can kill a cell. Why is it so lethal? EF2 contains diphthamide, an unusual amino acid residue that enhances the fidelity of codon shifting during translocation. Diphthamide is formed by a highly conserved complicated pathway that posttranslationally modifies histidine. The A fragment of the diphtheria toxin catalyzes the transfer of the ADP ribose unit of NAD⁺ to the diphthamide ring (Figure 30.31). This ADP ribosylation of a single side chain of EF2 blocks EF2′s capacity to carry out the translocation of the growing polypeptide chain. Protein synthesis ceases, accounting for the remarkable toxicity of diphtheria toxin.

Ricin is a small protein (65 kDa) found in the seeds of the castor oil plant, Ricinus communis (Figure 30.32). It is indeed a deadly molecule because as little as 500 μg is lethal for an adult human being, and a single molecule can inhibit all protein synthesis in a cell, resulting in cell death.

Ricin is a heterodimeric protein composed of a catalytic A chain joined by a single disulfide bond to a B chain. The B chain allows the toxin to bind to the target cell, and this binding leads to an endocytotic uptake of the dimer and the eventual release of the A chain into the cytoplasm. The A chain, an N-glycoside hydrolyase, cleaves adenine from a particular adenosine nucleotide on the 28S rRNA that is found in all eukaryotic ribosomes. Removal of the adenine base completely inactivates the ribosome by preventing the binding of elongation factors. Thus, ricin and diphtheria toxin both act by inhibiting protein-synthesis elongation; ricin does so by covalently modifying rRNA, and diphtheria toxin does so by covalently modifying the elongation factor.