Protein Synthesis: Termination of Polypeptide Synthesis Requires a Special Signal
Elongation continues until the ribosome adds the last amino acid coded by the mRNA. Termination, the fourth stage of polypeptide synthesis, is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA), immediately following the final coded amino acid. Mutations in a tRNA anticodon that allow an amino acid to be inserted at a termination codon are generally deleterious to the cell . In bacteria, once a termination codon occupies the ribosomal A site, three termination factors, or re lease factors—the proteins RF-1, RF-2, and RF-3— contribute to (1) hydrolysis of the terminal peptidyl tRNA bond; (2) release of the free polypeptide and the last tRNA, now uncharged, from the P site; and (3) dis sociation of the 70S ribosome into its 30S and 50S sub units, ready to start a new cycle of polypeptide synthesis (Fig. 1). RF-1 recognizes the termination codons UAG and UAA, and RF-2 recognizes UGA and UAA. Either RF-1 or RF-2 (depending on which codon is present) binds at a termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid. The release factors have domains thought to mimic the structure of tRNA, as shown for the elongation factor EF-G in Figure 27–25b. The specific function of RF-3 has not been firmly established, although it is thought to release the ribosomal subunit. In eukaryotes, a single release factor, eRF, recognizes all three termination codons.
Energy Cost of Fidelity in Protein Synthesis, Synthesis of a protein true to the information specified in its mRNA requires energy. Formation of each aminoacyl-tRNA uses two high-energy phosphate groups. An additional ATP is consumed each time an incorrectly activated amino acid is hydrolyzed by the deacylation activity of an aminoacyl-tRNA synthetase, as part of its proof-reading activity. A GTP is cleaved to GDP and Pi during the first elongation step, and another during the translocation step. Thus, on average, the energy derived from the hydrolysis of more than four NTPs to NDPs is re quired for the formation of each peptide bond of a polypeptide.
This represents an exceedingly large thermodynamic “push” in the direction of synthesis: at least 4 30.5 kJ/mol=122 kJ/mol of phosphodiester bond en ergy to generate a peptide bond, which has a standard free energy of hydrolysis of only about -21 kJ/mol. The net free-energy change during peptide bond synthesis is thus -101 kJ/mol. Proteins are information-containing polymers. The biochemical goal is not simply the formation of a peptide bond but the formation of a peptide bond between two specified amino acids. Each of the high-energy phosphate compounds expended in this process plays a critical role in maintaining proper align ment between each new codon in the mRNA and its as sociated amino acid at the growing end of the polypep tide. This energy permits very high fidelity in the biological translation of the genetic message of mRNA into the amino acid sequence of proteins.
Rapid Translation of a Single Message by Polysomes Large clusters of 10 to 100 ribosomes that are very active in protein synthesis can be isolated from both eukaryotic and bacterial cells. Electron micrographs show a fiber between adjacent ribosomes in the cluster, which is called a polysome . The connecting strand is a single molecule of mRNA that is being translated simultaneously by many closely spaced ribosomes, allowing the highly efficient use of the mRNA. In bacteria, transcription and translation are tightly coupled. Messenger RNAs are synthesized and trans lated in the same 5n 3direction. Ribosomes begin translating the 5end of the mRNA before transcription is complete . The situation is quite different in eukaryotic cells, where newly transcribed mRNAs must leave the nucleus before they can be translated. Bacterial mRNAs generally exist for just a few minutes before they are degraded by nucleases. In order to maintain high rates of protein synthesis, the mRNA for a given protein or set of proteins must be made continuously and translated with maximum efficiency. The short lifetime of mRNAs in bacteria allows a rapid cessation of synthesis when the protein is no longer needed.
FIGURE 1 Termination of protein synthesis in bacteria. Termi nation occurs in response to a termination codon in the A site. First, a release factor, RF (RF-1 or RF-2, depending on which termination codon is present), binds to the A site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypeptide. Finally, the mRNA, de acylated tRNA, and release factor leave the ribosome, and the ribosome dissociates into its 30S and 50S subunits.
