Protein Synthesis: Peptide Bonds Are Formed in the Elongation Stage
The third stage of protein synthesis is elongation. Again, our initial focus is on bacterial cells. Elongation requires (1) the initiation complex described above, (2) aminoacyl-tRNAs, (3) a set of three soluble cytosolic proteins called elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), and (4) GTP. Cells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added.
Elongation Step 1: Binding of an Incoming Aminoacyl-tRNA In the first step of the elongation cycle (Fig. 1), the appropriate incoming aminoacyl-tRNA binds to a com plex of GTP-bound EF-Tu. The resulting aminoacyl tRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex. The GTP is hydrolyzed and an EF-Tu–GDP complex is released from the 70S ribosome. The EF-Tu–GTP complex is regenerated in a process in volving EF-Ts and GTP. Elongation Step 2: Peptide Bond Formation A peptide bond is now formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosome. This occurs by the transfer of the initiating N-formylme thionyl group from its tRNA to the amino group of the second amino acid, now in the A site (Fig. 2). The-amino group of the amino acid in the A site acts as a nucleophile, displacing the tRNA in the P site to form the peptide bond. This reaction produces a dipeptidyl tRNA in the A site, and the now “uncharged” (deacylated) tRNAfMet remains bound to the P site. The tRNAs then shift to a hybrid binding state, with elements of each spanning two different sites on the ribosome, as shown in Figure 2. The enzymatic activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal sub unit. We now know that this reaction is catalyzed by the 23S rRNA, adding to the known catalytic repertoire of ribozymes. This discovery has interesting implications for the evolution of life .
FIGURE 1 First elongation step in bacteria: binding of the second aminoacyl-tRNA. The second aminoacyl-tRNA enters the A site of the ribosome bound to EF-Tu (shown here as Tu), which also contains GTP. Binding of the second aminoacyl-tRNA to the A site is ac companied by hydrolysis of the GTP to GDP and Pi and release of the EF-Tu–GDP complex from the ribosome. The bound GDP is released when the EF-Tu–GDP complex binds to EF-Ts, and EF-Ts is subsequently released when another molecule of GTP binds to EF-Tu. This recycles EF-Tu and makes it available to repeat the cycle.
FIGURE2 Second elongation step in bacteria: formation of the first peptide bond. The peptidyl transferase catalyzing this reaction is the 23S rRNA ribozyme. The N-formyl methionyl group is transferred to the amino group of the second aminoacyl-tRNA in the A site, forming a dipeptidyl-tRNA. At this stage, both tRNAs bound to the ribosome shift position in the 50S subunit to take up a hybrid binding state. The uncharged tRNA shifts so that its 3 and 5 ends are in the E site. Similarly, the 3 and 5 ends of the peptidyl tRNA shift to the P site. The anticodons remain in the A and P sites.
Elongation Step 3: Translocation In the final step of the elongation cycle, translocation, the ribosome moves one codon toward the 3end of the mRNA (Fig. 3a). This movement shifts the anticodon of the dipeptidyl tRNA, which is still attached to the second codon of the mRNA, from the A site to the P site, and shifts the de acylated tRNA from the P site to the E site, from where the tRNA is released into the cytosol. The third codon of the mRNA now lies in the A site and the second codon in the P site. Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the en ergy provided by hydrolysis of another molecule of GTP.
A change in the three-dimensional conformation of the entire ribosome results in its movement along the mRNA. Because the structure of EF-G mimics the structure of the EF-Tu–tRNA complex (Fig. 3b), EF-G can bind the A site and presumably displace the peptidyl-tRNA. The ribosome, with its attached dipeptidyl-tRNA and mRNA, is now ready for the next elongation cycle and attachment of a third amino acid residue. This process occurs in the same way as addition of the second residue (as shown in Figs 27–23, 27–24, and 27–25). For each amino acid residue correctly added to the growing polypeptide, two GTPs are hydrolyzed to GDP and Pi as the ribosome moves from codon to codon along the mRNA toward the 3end. The polypeptide remains attached to the tRNA of the most recent amino acid to be inserted. This association maintains the functional connection between the information in the mRNA and its decoded polypeptide output. At the same time, the ester linkage between this tRNA and the carboxyl terminus of the growing polypep tide activates the terminal carboxyl group for nucleophilic attack by the incoming amino acid to form a new peptide bond (Fig. 27–24). As the existing ester linkage between the polypeptide and tRNA is broken during peptide bond formation, the linkage between the poly peptide and the information in the mRNA persists, because each newly added amino acid is still attached to its tRNA. The elongation cycle in eukaryotes is quite simi lar to that in prokaryotes. Three eukaryotic elongation factors (eEF1α, eEF1βγ, and eEF2) have functions analogous to those of the bacterial elongation factors (EF-Tu, EF-Ts, and EF-G, respectively). Eukaryotic ribosomes do not have an E site; uncharged tRNAs are expelled directly from the P site.
Proofreading on the Ribosome The GTPase activity of EF Tu during the first step of elongation in bacterial cells (Fig. 27–23) makes an important contribution to the rate and fidelity of the overall biosynthetic process. Both the EF-Tu–GTP and EF-Tu–GDP complexes exist for a few milliseconds before they dissociate. These two intervals provide opportunities for the codon-anticodon interactions to be proofread. Incorrect aminoacyl-tRNAs normally dissociate from the A site during one of these periods. If the GTP analog guanosine 5-O-(3-thio triphosphate) (GTPS) is used in place of GTP, hydro lysis is slowed, improving the fidelity (by increasing the proofreading intervals) but reducing the rate of protein synthesis.
The process of protein synthesis (including the characteristics of codon-anticodon pairing already described) has clearly been optimized through evolution to balance the requirements of both speed and fidelity. Improved fidelity might diminish speed, whereas increases in speed would probably compromise fidelity. Note that the proofreading mechanism on the ribosome establishes only that the proper codon-anticodon pairing has taken place. The identity of the amino acid at tached to a tRNA is not checked on the ribosome. If a tRNA is successfully aminoacylated with the wrong amino acid (as can be done experimentally), this incorrect amino acid is efficiently incorporated into a protein in response to whatever codon is normally recognized by the tRNA.
FIGURE 3 Third elongation step in bacteria: translocation. (a) The ribosome moves one codon toward the 3end of the mRNA, using energy provided by hydrolysis of GTP bound to EF-G (translocase). The dipeptidyl-tRNA is now entirely in the P site, leaving the A site open for the incoming (third) aminoacyl-tRNA. The uncharged tRNA dissociates from the E site, and the elongation cycle begins again. (b) The structure of EF-G mimics the structure of EF-Tu complexed with tRNA. Shown here are (left) EF-Tu complexed with tRNA (green) (PDB ID 1B23) and (right) EF-G complexed with GDP (red) (PDB ID 1DAR). The carboxyl-terminal part of EF-G (dark gray) mimics the structure of the anticodon loop of tRNA in both shape and charge distribution.
