Peptide bond formation
aminoacyl-tRNA is brought into the ribosome as a ternary complex in association with EF1a-GTP and becomes bound to the A site, so named because it is where aminoacylated tRNAs bind (step 1). If the anticodon of the incoming (second) aminoacyl-tRNA correctly base-pairs with the second codon of the mRNA, the GTP in the associated EF1a-GTP is hydrolyzed. The hydrolysis of GTP promotes a conforma-tional change in the ribosome that leads to tight binding of the aminoacyl-tRNA in the A site and release of the resulting EF1a-GDP complex (step 2). This conformational change also positions the aminoacylated 3' end of the tRNA in the A site in close proximity to the 3' end of the Met-tRNA(Met in the P site. GTP hydrolysis, and hence tight binding, does not occur if the anticodon of the incoming aminoacyl-tRNA cannot base-pair with the codon at the A site. In this case, the ternary complex diffuses away, leaving an empty A site that can associate with other aminoacyltRNA-EF1a-GTP complexes until a correctly base-paired tRNA is bound. This phenomenon contributes to the fidelity with which the correct aminoacyl-tRNA is loaded into the A site.
With the initiating Met-tRNA(Met at the P site and the second aminoacyl-tRNA tightly bound at the A site, the a amino group of the second amino acid reacts with the "activated" (ester-linked) methionine on the initiator tRNA, forming a peptide bond (Figure 4-26, step 3; see Figures 4-19 and 4-21). This peptidyltransferase reaction is catalyzed by the large rRNA, which precisely orients the interacting atoms, permitting the reaction to proceed. The catalytic ability of the large rRNA in bacteria has been demonstrated by carefully removing the vast majority of the protein from large ribosomal subunits. The nearly pure bacterial 23S rRNA can catalyze a peptidyltransferase reaction between analogs of aminoacylated-tRNA and peptidyl-tRNA. Further support for the catalytic role of large rRNA in protein synthesis comes from crystallographic studies showing that no proteins lie near the site of peptide bond synthesis in the crystal structure of the bacterial large subunit.
Following peptide bond synthesis, the ribosome is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2-GTP. As a result of translocation, tRNAiMet, now without its activated methi-onine, is moved to the E (exit) site on the ribosome; concurrently, the second tRNA, now covalently bound to a dipeptide (a peptidyl-tRNA), is moved to the P site (Figure 4-26, step 4). Translocation thus returns the ri-bosome conformation to a state in which the A site is open and able to accept another aminoacylated tRNA complexed with EF1a-GTP, beginning another cycle of chain elongation.
Repetition of the elongation cycle depicted in Figure 4-26 adds amino acids one at a time to the C-terminus of the growing polypeptide as directed by the mRNA sequence until a stop codon is encountered. In subsequent cycles, the conformational change that occurs in step 2 ejects the unacylated tRNA from the E site. As the nascent polypeptide chain becomes longer, it threads through a channel in the large ribosomal subunit, exiting at a position opposite the side that interacts with the small subunit (Figure 4-27).
The locations of tRNAs bound at the A, P, and E sites are visible in the recently determined crystal structure of the bacterial ribosome (Figure 4-28). Base pairing is also apparent between the tRNAs in the A and P sites with their respective codons in mRNA (see Figure 4-28, inset). An RNA-RNA hybrid of only three base pairs is not stable under physio-
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