30.1 Protein Synthesis Requires the Translation of Nucleotide Sequences into Amino Acid Sequences

Protein synthesis is called translation because information present as a nucleic acid sequence is translated into a different language, the sequence of amino acids in a protein. This complex process is mediated by the coordinated interplay of more than a hundred macromolecules, including mRNA, rRNAs, tRNAs, aminoacyl-tRNA synthetases, and protein factors. Given that a protein typically comprises from 100 to 1000 amino acids, the frequency at which an incorrect amino acid is incorporated in the course of protein synthesis must be less than 10⁻⁴. Transfer RNAs are the adaptors that make the link between a nucleic acid and an amino acid. These molecules, single chains of about 80 nucleotides, have an L-shaped structure.

30.2 Aminoacyl Transfer RNA Synthetases Read the Genetic Code

Each amino acid is activated and linked to a specific transfer RNA by an enzyme called an aminoacyl-tRNA synthetase. The synthetase links the carboxyl group of an amino acid to the 2′- or 3′-hydroxyl group of the adenosine unit of a CCA sequence at the 3′ end of the tRNA by an ester linkage. There is at least one specific aminoacyl-tRNA synthetase and at least one specific tRNA for each amino acid. A synthetase utilizes both the functional groups and the shape of its cognate amino acid to prevent the attachment of an incorrect amino acid to a tRNA. Some synthetases have a separate active site at which incorrectly linked amino acids are removed by hydrolysis. A synthetase recognizes the anticodon, the acceptor stem, and sometimes other parts of its tRNA substrate. By specifically recognizing both amino acids and tRNAs, aminoacyl-tRNA synthetases implement the instructions of the genetic code.

The codons of messenger RNA recognize the anticodons of transfer RNAs rather than the amino acids attached to the tRNAs. A codon on mRNA forms base pairs with the anticodon of the tRNA. Some tRNAs are recognized by more than one codon because pairing of the third base of a codon is less crucial than that of the other two (the wobble mechanism). There are two evolutionary distinct classes of synthetases, each recognizing 10 amino acids. The two classes recognize opposite faces of tRNA molecules.

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30.3 The Ribosome Is the Site of Protein Synthesis

Protein synthesis takes place on ribosomes—ribonucleoprotein particles (about two-thirds RNA and one-third protein) consisting of large and small subunits. In E. coli, the 70S ribosome (2500 kDa) is made up of 30S and 50S subunits. The 30S subunit consists of 16S ribosomal RNA and 21 different proteins; the 50S subunit consists of 23S and 5S rRNA and 34 different proteins. The ribosome includes three sites for tRNA binding called the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site.

Protein synthesis takes place in three phases: initiation, elongation, and termination. In bacteria, mRNA, formylmethionyl-tRNA_f (the special initiator tRNA that recognizes AUG), and a 30S ribosomal subunit come together with the assistance of initiation factors to form a 30S initiation complex. A 50S ribosomal subunit then joins this complex to form a 70S initiation complex, in which fMet-tRNA_f occupies the P site of the ribosome.

Elongation factor Tu delivers the appropriate aminoacyl-tRNA to the ribosome’s A site as an EF-Tu–aminoacyl-tRNA–GTP ternary complex. EF-Tu serves both to protect the aminoacyl-tRNA from premature cleavage and to increase the fidelity of protein synthesis by ensuring that the correct anticodon–codon pairing has taken place before hydrolyzing GTP and releasing aminoacyl-tRNA into the A site. A peptide bond is formed when the amino group of the aminoacyl-tRNA nucleophilically attacks the ester linkage of the peptidyl-tRNA. On peptide-bond formation, the tRNAs and mRNA are translocated for the next cycle to begin. The deacylated tRNA moves to the E site and then leaves the ribosome, and the peptidyl-tRNA moves from the A site into the P site. Elongation factor G uses the free energy of GTP hydrolysis to drive translocation. Protein synthesis is terminated by release factors, which recognize the termination codons UAA, UGA, and UAG and cause the hydrolysis of the ester bond between the polypeptide and tRNA.

30.4 Eukaryotic Protein Synthesis Differs from Bacterial Protein Synthesis Primarily in Translation Initiation

The basic plan of protein synthesis in eukaryotes is similar to that in bacteria, but there are some significant differences between them. Eukaryotic ribosomes (80S) consist of a 40S small subunit and a 60S large subunit. The initiating amino acid is again methionine, but it is not formylated. The initiation of protein synthesis is more complex in eukaryotes than in bacteria. In eukaryotes, the AUG closest to the 5′ end of mRNA is nearly always the start site. The 40S ribosome finds this site by binding to the 5′ cap and then scanning the RNA until AUG is reached. The regulation of translation in eukaryotes provides a means for regulating gene expression.

30.5 A Variety of Antibiotics and Toxins Can Inhibit Protein Synthesis

Many clinically important antibiotics function by inhibiting protein synthesis. All steps of protein synthesis are susceptible to inhibition by one antibiotic or another. Diphtheria toxin inhibits protein synthesis by covalently modifying an elongation factor, thereby preventing elongation. Ricin, a toxin from castor beans, inhibits elongation by removing a crucial adenine residue from rRNA.

30.6 Ribosomes Bound to the Endoplasmic Reticulum Manufacture Secretory and Membrane Proteins

Proteins contain signals that determine their ultimate destination. The synthesis of all proteins begins on free ribosomes in the cytoplasm. In eukaryotes, protein synthesis continues in the cytoplasm unless the nascent chain contains a signal sequence that directs the ribosome to the endoplasmic reticulum. Amino-terminal signal sequences consist of a hydrophobic segment of 9 to 12 residues preceded by a positively charged amino acid. The signal-recognition particle, a ribonucleoprotein assembly, recognizes signal sequences and brings ribosomes bearing them to the ER. A GTP–GDP cycle releases the signal sequence from SRP and then detaches SRP from its receptor. The nascent chain is then translocated across the ER membrane. Proteins are transported throughout the cell in transport vesicles.

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