BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 27. PROTEIN SYNTHESIS

27.18. Protein Synthesis is Terminated by Release Factors

If the A site of the ribosome is occupied by UAA, UGA, or UAG codons, binding of aminoacyl-tRNA typically does not occur. Normal Cells do not contain tRNAs with anticodons complementary to termination signals. Instead, these signals are recognized by protein release factors. One such release factor, RF-1, recognizes UAA or UAG codons, whereas a second release factor, RF-2, recognizes UAA or UGA. Thus, Proteins are capable of recognizing trinucleotide sequences with high Specificity.

The binding of a release factor to the termination codon in the A site somehow activates peptidyl transferase, which then hydrolyzes the bond between the polypeptide and the tRNA in the P site. The release factor alters the specificity of peptidyl transferase such that H₂O, rather than an amino group, acts as the acceptor for the activated peptidyl moiety. The polypeptide chain then leaves the ribosome. The 70S ribosome dissociates into 30S and 50S subunits, preparing for the synthesis of a new protein molecule.

27.19. Many Proteins Undergo Post-Translational Modification

Many Polypeptides generated during mRNA Translation are not yet final products; they may subsequently be modified in various ways.

1. The formyl group at the N-terminus of bacterial proteins is hydrolyzed by deformylase. An aminopeptidase may then cleave one or more N-terminal residues. In both PROKARYOTES AND EUKARYOTES, the terminal Methionine is sometimes cleaved while the Synthesis of the rest of the polypeptide chain is still underway.

2. Oxidation of two Cysteine residues can lead to The formation of Disulfide Bonds.

3. The side chains of Certain Amino Acids can be specifically modified. For instance, Certain Proline and Lysine residues in Collagen are hydroxylated. Glycoproteins are formed by attaching sugars to the side chains of asparagine, Serine, and Threonine. Some proteins are phosphorylated. Prosthetic groups, such as Lipoic Acid, are covalently attached to Enzymes.

4. Polypeptide chains may undergo specific Cleavage, such as The conversion of procollagen to collagen and proinsulin to Insulin. Translation of polyoma virus mRNA yields a very long polypeptide chain that is hydrolyzed to produce multiple proteins.

27.20. Streptomycin Inhibits Initiation and Causes Misreading of Messenger RNA

As we have seen, Antibiotics are exceptionally valuable tools in biochemical research because the action of many of them is highly specific. For example, rifampicin is a potent inhibitor of RNA Synthesis initiation (Section 25.18). Numerous antibiotics are known to inhibit Protein Synthesis (Table 27.3), and for several of them, the MECHANISM OF ACTION has been established. Streptomycin, a strongly basic trisaccharide, prevents the binding of formylmethionyl-tRNA to Ribosomes, thereby disrupting the proper initiation of protein synthesis. In addition, streptomycin induces mRNA misreading. When poly(U) is used as a template, isoleucine (AUU) is incorporated alongside phenylalanine (UUU). The site of streptomycin action on the ribosome was determined through reconstitution experiments involving ribosomal components from streptomycin-sensitive and streptomycin-resistant Bacteria. Such bacterial strains differ by a mutation in a single Gene. How do these ribosomes differ? Because ribosomes can be dissociated and reconstituted in vitro, it became possible to determine whether the determinant of streptomycin sensitivity resides in the 50S or the 30S subunit. Hybrid ribosomes composed of 50S subunits from resistant bacteria and 30S subunits from sensitive bacteria proved to be sensitive to streptomycin, whereas the reciprocal combination yielded resistant ribosomes. This experiment demonstrated that the determinant for streptomycin sensitivity is localized in the 30S subunit. A subsequent experiment showed that sensitivity to streptomycin is conferred by a specific protein of the 30S subunit rather than the 16S rRNA molecule. Finally, after a series of complex experiments, it was found that streptomycin sensitivity is determined by a single protein component of the 30S subunit, S12.

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Table 27.3. Antibiotic Inhibitors of Protein Synthesis

27.21. Puromycin Causes Premature Chain Termination by Mimicking Aminoacylated Transfer RNA

The antibiotic puromycin inhibits protein synthesis by prematurely releasing nascent polypeptide chains before their synthesis is complete. Puromycin is an analog of the terminal region of aminoacyl-tRNA, specifically aminoacyl adenosine (Fig. 27.24). It binds to the A site of the ribosome and blocks the binding of aminoacyl-tRNA. Furthermore, puromycin contains an α-amino group. Like the amino group of aminoacyl-tRNA, this amino group forms a peptide bond with the carboxyl group of the growing peptide chain in a reaction catalyzed by peptidyl transferase. The resulting product is a peptide with a covalently attached puromycin residue at its carboxyl terminus. Peptidyl-puromycin then dissociates from the ribosome. Puromycin has been instrumental in studying the functional states of ribosomes. METABOLISM/2.html">THE CONCEPT OF A and P sites emerged from experiments using puromycin to determine the localization of peptidyl-tRNA. When peptidyl-tRNA is located in the A site (prior to translocation), it cannot react with puromycin.

Fig. 27.24. The Structure of puromycin resembles the aminoacylated end of aminoacyl-tRNA