Figure 6–52 Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a four-subunit enzyme) removes the tRNA intron (dark blue, bottom). A second enzyme, a multifunctional tRNA ligase (not shown), then joins the two tRNA halves together. (Courtesy of Hong Li, Christopher Trotta, and John Abelson; PDB code: 2A9L.) ribose HHNNNNNCH3CH3ribose HNNOHHHHribose HNNSOHHtwo methyl groups added to G (N,N-dimethyl G) two hydrogens added to U (dihydro U) ribose HNNNNHHOdeamination of A (inosine) sulfur replaces oxygen in U (4-thiouridine) P P P P has an anticodon that matches the amino acid. A second enzyme then chemically modifies each “incorrectly” attached amino acid so that it now corresponds to the anticodon displayed by its covalently linked tRNA.
The synthetase-catalyzed reaction that attaches the amino acid to the 3ʹend of the tRNA is one of many reactions coupled to the energy-releasing hydrolysis of ATP (see pp. 64–65), and it produces a high-energy bond between the tRNA and the amino acid. The energy of this bond is used at a later stage in protein synthesis to link the amino acid covalently to the growing polypeptide chain.
The aminoacyl-tRNA synthetase enzymes and the tRNAs are equally important in the decoding process (Figure 6–56). This was established by an experiment in
Figure 6–54 Amino acid activation by synthetase enzymes. An amino acid is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme in two steps. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl group directly to AMP, forming an adenylated amino acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3ʹ end of the tRNA molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule. The synthetase enzyme is not shown in this diagram.
Figure 6–53 A few of the unusual nucleotides found in tRNA molecules.
These nucleotides are produced by covalent modification of a normal nucleotide after it has been incorporated into an RNA chain. Two other types of modified nucleotides are shown in Figure 6–41. In most tRNA molecules, about 10% of the nucleotides are modified (see Figure 6–50). As shown in Figure 6–51, inosine is sometimes present at the wobble position in the tRNA anticodon.
which one amino acid (cysteine) was chemically converted into a different amino acid (alanine) after it already had been attached to its specific tRNA. When such “hybrid” aminoacyl-tRNA molecules were used for protein synthesis in a cell-free system, the wrong amino acid was inserted at every point in the protein chain where that tRNA was used. Although, as we shall see, cells have several quality control mechanisms to avoid this type of mishap, the experiment did establish that the genetic code is translated by two sets of adaptors that act sequentially. Each matches one molecular surface to another with great specificity, and it is their combined action that associates each sequence of three nucleotides in the mRNA molecule—that is, each codon—with its particular amino acid.