Development of tRNA synthetases and connection to genetic code and disease

Abstract

The genetic code is established by the aminoacylation reactions of aminoacyl tRNA synthetases, where amino acids are matched with triplet anticodons imbedded in the cognate tRNAs. The code established in this way is so robust that it gave birth to the entire tree of life. The tRNA synthetases are organized into two classes, based on their active site architectures. The details of this organization, and other considerations, suggest how the synthetases evolved by gene duplications, and how early proteins may have been statistical in nature, that is, products of a primitive code where one of several similar amino acids was used at a specific position in a polypeptide. The emergence of polypeptides with unique, defined sequences--true chemical entities--required extraordinary specificity of the aminoacylation reaction. This high specificity was achieved by editing activities that clear errors of aminoacylation and thereby prevent mistranslation. Defects in editing activities can be lethal and lead to pathologies in mammalian cells in culture. Even a mild defect in editing is casually associated with neurological disease in the mouse. Defects in editing are also mutagenic in an aging organism and suggest how mistranslation can lead to mutations that are fixed in the genome. Thus, clearance of mischarged tRNAs by the editing activities of tRNA synthetases was essential for development of the tree of life and has a role in the etiology of diseases that is just now being understood.

Figure 1.

The tree of life. The tree was made possible by a robust genetic code that was needed for the transition from the RNA world to the theatre of proteins.

Figure 2.

Classes of aminoacyl tRNA synthetases and their implications for development of the genetic code. (A) The two major classes can be organized into subclasses that hold enzymes that are most closely related to each other in their sequences. Significantly, the subclasses also group tRNA synthetases according to their amino acid chemical types. Data adapted from Ribas de Pouplana and Schimmel (2004a). (B) Evolution of the two classes of synthetases from an ancestral gene where complementary strands of the gene code for, respectively, class I (red) and II (green) proteins. Subsequent gene duplications gave rise to the subclasses (depicted in A). Data adapted from Ribas de Pouplana and Schimmel (2004b). (C) The ancient catalytic domains of the synthetases can dock to opposite sides of the tRNA acceptor stems (as shown in B), in a way that is highly specific and that recapitulates the organization of subclasses in A.

Figure 3.

Illustration of distinct active sites for amino acid activation and editing. The two sites, in the example of IleRS, are separated by ∼25 Å. A misactivated amino acid, such as valine, is translocated from the site for activation to the site for editing, where it is cleared. This translocation requires tRNA. The two distinct sites were first identified by chemical and genetic methods. Adapted from Schimmel (2008) (reprinted with permission from the American Society for Biochemistry and Molecular Biology ©2008).

Figure 4.

Analysis of editing-defective murine T535P ValRS. (A) The mutation does not affect aminoacylation (left) but severely attenuates the clearance of Thr-tRNA^Val (right). (B) Highly conserved sequences of the region around T535P of murine ValRS and the placement of the mutation in the structure of the cocrystal of ValRS with a threonyl-AMP analog. (C) Visualization of the activation of caspase-3, using a fluorescence assay, in mouse 3T3 fibroblasts harboring the editing-defective T535P ValRS. (D) A simple biosensor to detect by fluorescence the substitution of Thr for Val at codon 65 in the enhanced form of green fluorescent protein (EGFP). Fluorescence is greatly enhanced by the insertion of Thr (from Thr-tRNA^Val) instead of Val. Adapted from Nangle et al. (2006) (reprinted with permission from Elsevier ©2006).

Figure 5.

The G3:U70 base pair marks a tRNA for acceptance of alanine. G:U recognition occurs with determinants in the catalytic domain of AlaRS. Remarkably, the editing domain is also sensitive to the same G:U base pair. Thus, two distinct domains in AlaRSs recognize the same base pair.

Figure 6.

In aging bacteria that are grown in the presence of ciprofloxin, mutations accumulate in enzymes needed to relax supercoiling of DNA. These mutations are diagnostic for induction of the SOS response and the error-prone DNA repair systems. Adapted from Bacher and Schimmel (2007) (reprinted with permission from The National Academy of Sciences ©2007).