Several mechanisms working together ensure that an aminoacyl-tRNA synthetase links the correct amino acid to each tRNA. Most synthetase enzymes select the correct amino acid by a two-step mechanism. The correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favored over the other 19; in particular, amino acids larger than the correct one are excluded from the active site. However, accurate discrimination between two similar amino acids, such as isoleucine and valine (which differ by only a methyl linkage of amino acid to tRNA
Figure 6–55 The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose. Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an amino acid held in this way is said to be activated.
Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3ʹ end of the tRNA (see Figure 6–50).
Actual structure corresponding to the boxed region in (A). There are two major classes of synthetase enzymes: one links the amino acid directly to the 3ʹ-OH group of the ribose, and the other links it initially to the 2ʹ-OH group. In the latter case, a subsequent transesterification reaction shifts the amino acid to the 3ʹ position. As in Figure 6–54, the “R group” indicates the side chain of the amino acid.
Figure 6–56 The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be incorporated into a protein chain (Movie 6.6). In the sequence of events shown, the amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA.
group), is very difficult to achieve in a single step. A second discrimination step occurs after the amino acid has been covalently linked to AMP (see Figure 6–54): when tRNA binds, the synthetase tries to force the adenylated amino acid into a second editing pocket in the enzyme. The precise dimensions of this pocket exclude the correct amino acid, while allowing access by closely related amino acids. In the editing pocket, an amino acid is removed from the AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed) by hydrolysis. This hydrolytic editing, which is analogous to the exonucleolytic proofreading by DNA polymerases, increases the overall accuracy of tRNA charging to approximately one mistake in 40,000 couplings (Figure 6–57).
The tRNA synthetase must also recognize the correct set of tRNAs, and extensive structural and chemical complementarity between the synthetase and the tRNA allows the synthetase to probe various features of the tRNA (Figure 6–58). Most tRNA synthetases directly recognize the matching tRNA anticodon; these synthetases contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and charge to a nucleotide in the anticodon. For other synthetases, the nucleotide sequence of the amino acid-accepting arm (acceptor stem) is the key recognition determinant. In most cases, however, the synthetase “reads” the nucleotides at several different positions on the tRNA.
Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain