Termination occurs when one of the three termination codons moves into the A site. These codons are recognized in E. coli by release factors: RF-1, which recognizes UAA and UAG, and RF-2, which recognizes UGA and UAA. The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP, then causes the release of RF-1 or RF-2 as GTP is hydrolyzed (see Fig. 32.13). [Note: Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, functions like the prokaryotic RF-3. See Figure 32.14 for a summary of the factors used in translation.] The steps in prokaryotic protein synthesis, as well as some antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide. [Note: In prokaryotes, ribosome recycling factors mediate separation of the subunits. In eukaryotes, eRF and ATP hydrolysis are required.]
D. Translation regulation
Gene expression is most commonly regulated at the transcriptional level, but translation may also be regulated. An important mechanism by which this is achieved in eukaryotes is by covalent modification of eIF-2: Phosphorylated eIF-2 is inactive (see p. 476). In both eukaryotes and prokaryotes, regulation can also be achieved through proteins that bind mRNA and inhibit its use by blocking translation.
E. Protein folding
Proteins must fold to assume their functional, native state. Folding can be spontaneous (as a result of the primary structure) or facilitated by proteins known as chaperones (see p. 20).
F. Protein targeting
Although most protein synthesis in eukaryotes is initiated in the cytoplasm, many proteins perform their functions within subcellular organelles or outside of the cell. Such proteins normally contain amino acid sequences that direct the proteins to their final locations. For example, secreted proteins are targeted during synthesis (cotranslational targeting) to the RER by the presence of an N-terminal hydrophobic signal sequence. The sequence is recognized by the signal recognition particle (SRP), a ribonucleoprotein that binds the ribosome, halts elongation, and delivers the ribosome–peptide complex to an RER membrane channel (the translocon) via interaction with the SRP receptor. Translation resumes, the protein enters the RER lumen, and its signal sequence is cleaved (Fig. 32.15). The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an internal, short, basic nuclear localization signal; mitochondrial matrix proteins that contain an N-terminal, amphipathic, α-helical mitochondrial entry sequence; and peroxisomal proteins that contain a C-terminal tripeptide signal.
VI. CO-AND POSTTRANSLATIONAL MODIFICATIONS
Many polypeptides are covalently modified, either while they are still attached to the ribosome (cotranslational) or after their synthesis has been completed (posttranslational). These modifications may include removal of part of the translated sequence or the covalent addition of one or more chemical groups required for protein activity.
A. Trimming