Aminoacyl tRNA Synthetases Part 1 (Molecular Biology)

The aminoacyl tRNA synthetases catalyze reactions that establish the rules of the genetic code. For this reason, there is great interest in these enzymes and their evolutionary development, which is thought to be closely connected to the establishment of the code. Research on the synthetases has led to the concept of an operational RNA code for amino acids that is imbedded in the acceptor stems of transfer RNA (tRNA) (1). The operational RNA code may have played an important role in the assembly of the genetic code and in the overall design of tRNA synthetases.

In the flow of genetic information, messenger RNA (mRNA) is transcribed from DNA, and the mRNA, in turn, is the template for protein synthesis (Fig. 1). The triplet codons of mRNA interact with the anticodons of tRNA through complementary base pairing. Amino acids joined to tRNA are incorporated into the growing polypeptide chain. The algorithm of the genetic code relates each amino acid to a specific trinucleotide codon. The triplet associated with a particular amino acid is determined in the aminoacylation reaction, where a given amino acid is linked to a tRNA bearing the anticodon trinucleotide that corresponds to that amino acid. These aminoacylation reactions are catalyzed by aminoacyl tRNA synthetases.

Figure 1. Flow of genetic information. Messenger RNA is synthesized from DNA. The mRNA has a string of trinucleotide codons that are translated into a polypeptide whose amino acid sequence is determined by the codons, according to the rules of the genetic code. The amino acid that is inserted into the polypeptide is determined by the codon-anticodon interaction with the tRNA that bears the amino acid corresponding to the particular anticodon. Therefore, the genetic code is determined by the linking of a particular amino acid with a particular anticodon triplet within a tRNA. The joining of amino acids to tRNA is catalyzed by aminoacyl tRNA synthetases. Thus, the genetic code is determined at the biochemical level in the aminoacylation reaction.

1. Aminoacylation Reaction and the Genetic Code

For most tRNA synthetases, aminoacylation is carried out in a two-step reaction:

In the first reaction, the enzyme, E, uses ATP to activate an amino acid, AA, to yield the firmly bound aminoacyladenylate (AA-AMP). In the second step, the activated amino acid is transferred to the 3′-end of the tRNA, where it is connected by an ester linkage to the 2′- or 3′-hydroxyl group (after initial attachment, the amino acid can migrate back and forth between the 2′ and the 3′ positions). While most synthetases can carry out amino acid activation in the absence of tRNA, there are a few exceptions (such as glutaminyl-, glutamyl-, and argininyl-tRNA synthetases) that require the presence of the cognate tRNA for amino acid activation.

Because each of the 20 natural amino acids used in protein synthesis has a corresponding, or cognate, aminoacyl tRNA synthetase, there are 20 of these enzymes in each cellular compartment where proteins are synthesized. Each of them must distinguish its amino acid from all others and, at the same time, recognize the cognate tRNA that bears the anticodon corresponding to that amino acid. In prokaryotes and in the cytoplasm of eukaryotes, there is typically one tRNA synthetase for each amino acid (eukaryotic mitochondria have an additional set of synthetases that are essential for mitochondrial protein synthesis). However, the degeneracy of the genetic code means that there are 61 trinucleotides coding for the 20 amino acids. Reading these 61 triplets requires more than just 20 tRNAs. As a consequence, there are multiple tRNA isoacceptors for many of the synthetases. The synthetases for a particular amino acid must, therefore, recognize and aminoacylate all tRNA isoacceptors for that amino acid. This consideration in itself has important implications.

For example, the codons for serine are sixfold degenerate. In order to read these six codons, the serine tRNA isoacceptors must collectively permute all three anticodon nucleotides. Thus, for a single seryl-tRNA synthetase to aminoacylate all these tRNA isoacceptors, the anticodon is not suitable for discrimination. Direct experiments in vitro and the X-ray crystallography structure of the seryl-tRNA synthetase-tRNA complex have demonstrated that, in fact, seryl-tRNA synthetase does not contact the anticodon trinucleotide (2). This observation, and others described below, showed that, for at least some amino acids, the relationship between an amino acid and the triplet of the genetic code is not direct.

2. Classes of Aminoacyl tRNA Synthetases

The synthetases are heterogeneous in quaternary structures and subunit sizes, and this heterogeneity obscured more fundamental relationships between these enzymes. For example, in Escherichia coli, the quaternary structures of synthetases include a, a2, a4, and a2b2 (3). Subunit sizes vary from 303 to 951 amino acid residues (4). The 20 aminoacyl tRNA synthetases are now known to be divided into two classes of 10 enzymes each (Table 1) (8, 9). These classes are based on conserved sequence motifs and the structural architecture of the catalytic domains (8, 9). The classification is also based on the fact that the site of initial amino acid attachment on the tRNA differs between the two classes (8). The classes appear to be fixed in evolution, because there is no example of an enzyme switching classes depending on the organism to which it belongs. Thus, the two classes may have developed early in evolution.

Table 1. Classes of Aminoacyl tRNA Synthetases

Gram-positive bacteria, plant chloroplasts, and animal mitochondria have been shown to have less than 20 tRNA synthetases. Instead, glutamyl-tRNA synthetase catalyzes attachment of glutamic acid to both tRNAGlu and tRNAGln and, similarly, aspartyl-tRNA synthetases catalyzes attachment of aspartate to both tRNAAsp and tRNAAsn. An amidotransferase then catalyzes the amidation of Glu-tRNAGln to give Gln-tRNAGln, and, likewise, amidation of Asp-tRNAAsn gives Asn-tRNAAsn (5-7).

2.1. Class I

These enzymes are usually monomers and are characterized by an architecture that is similar to that seen in dehydrogenases and other nucleotide-binding proteins. This structural motif is a Rossmann nucleotide-binding fold, which consists of alternating b-strands and a-helices (Fig. 2) (10-12). In the case of class I tRNA synthetases, the fold is divided into two b3a2 halves to give an overall b6a4 structure. In this structure, the b-strands are arranged in parallel. A polypeptide of variable length, designated as connective polypeptide 1 (CP1), links together the two halves of the active site (13). In some class I enzymes, this insertion plays a role in translational editing. It also contains some of the residues for binding the synthetase to the tRNA acceptor helix (14).

Figure 2. Design of a class I tRNA synthetase. The nucleotide-binding fold of class I tRNA synthetases consists of alten (cylinders) that form a b6a4 structure. A two-dimensional spatial arrangement of these elements is shown at the top, and ; below. A second domain of variable size occurs after the nucleotide-binding fold. The fold is split into two b^ halves b polypeptide 1 (CPI). A second, smaller insertion (CP2) splits the second half of the fold. Two sequence elements were us enzymes. These are known as the 12-residue signature sequence, which ends in the HIGH tetrapeptides (10, 11) and as t locations in the schematic structure are shown near the label signature sequence and KMSKS. By way of example, an ali sequences of the 10 class I E. coli enzymes is shown beneath the schematic figures. Similar alignments can be made for < throughout evolution.

The nucleotide-binding fold contains the site for adenylate synthesis. This catalytic domain may be identified by two characteristic sequence motifs, without any knowledge of three-dimensional structure. One motif is the 11-amino acid element known as the signature sequence, which ends in the sequence-His-Ile-Gly-His, or HIGH in one-letter code (10, 11). This element is located in the first half of the nucleotide-binding fold at the end of the first b-strand and the beginning of the first a-helix. It was designated as a signature sequence because it served as a clear signature for a subgroup of related synthetases, before many crystal structures were determined. The second element is the KMSKS motif, located in the second half of the nucleotide-binding fold (15). These elements are critical parts of the active site.

2.2. Class II

The class II enzymes are mostly a2 dimers. The active sites of class II enzymes have a completely different architecture that harbors three characteristic sequence motifs. The structure consists of a seven-stranded antiparallel b-sheet with three a-helices (9, 16-18) (Fig. 3) (8, 9, 19). The three characteristic sequence motifs are known as motifs 1, 2, and 3. The sequences of these motifs are highly degenerate (8, 9). They consist of a helix-loop-strand, strand-loop-strand, and strand-helix, respectively. All three of these motifs form part of the active site.

Figure 3. Design of a class II tRNA synthetase. The seven-stranded b-sheet with three a-helices is shown. The variable-s line that may occur on either the N- or the C-terminal side of the class-defining domain. Three characteristic sequence m and are distinguished in this illustration by their different shadings. These motifs are highly degenerate in sequence and c strand-loop-strand (motif 2), and strand-helix. The locations of these motifs in the class-defining domain are shown. Ar motifs for E. coli tRNA synthetases is also shown (8, 19). Note the high degeneracy of these sequence motifs, especially sequence elements in class I enzymes (Fig. 2).

2.3. Amino Acid Attachment

The site of initial amino acid attachment for class I enzymes is the 2′-hydroxyl, whereas the 3′-hydroxyl is used by class II enzymes (20). This distinction is now understood to result from a difference in the ways that the two enzymes approach the end of the tRNA. In particular, class I enzymes approach the end of the tRNA acceptor helix from the minor groove side, while class II enzymes approach from the major groove side (21).