10.3.1—
Amino Acid Activation and Aminoacyl-tRNA Synthesis

This energy-requiring process is accomplished in the cytosol by enzymes called aminoacyl-tRNA synthetases or ligases. It occurs as a two-stage reaction, both stages being catalysed by the same aminoacyl-tRNA synthetase enzyme (Fig. 10.4). The product of the reaction, aminoacyl-tRNA molecules, contain in the

Figure 10.4
The reactions of aminoacyl-tRNA synthesis. (In the cell a pyrophosphatase
acts on the pyrophosphate formed to make reaction (a) irreversible in practice).

aminoacyl-tRNA bond, sufficient energy for the subsequent formation of peptide bonds to occur spontaneously and, in addition, the anticodon triplet of bases of the tRNA ensures that the amino acid will be located at a particular residue position in the amino acid sequence of the protein product. This reaction, therefore, controls the proportion of free to aminoacylated-tRNAs in the cytoplasm, and theoretically therefore, could affect the rate and/or type of protein being synthesized.

10.3.1.1—
tRNA

The code words (codons) of the genetic code were first established for E. coil. This work showed that there were 61 codons shared between twenty protein amino acids, i.e. there is more than one codon for some amino acids and these can attach to more than one tRNA species. Chemically different tRNA species which can be acylated by the same amino acid are called isoacceptor tRNAs.

However, it was soon realized that not every codon has a corresponding tRNA with a specific anticodon and that some tRNAs recognize more than one codon. Crick's 'wobble hypothesis' (1966) to account for this, proposed alternative base-pairing to occur between the base in the third position of the mRNA codon and the corresponding base in the tRNA anticodon. The proposed rules are set out in Fig. 10.5. Thus, inosine in the 5' position in the anticodon of an

Figure 10.5
The basis of the 'wobble' hypothesis. (X =
Any nucleotide; I = Inosine; G = Guanine;
U = Uridine; A = Adenine; C = Cytosine;)
(From Boulter et al.  (Biol. Rev.  47,  113–75, 1972.))

aminoacyl-tRNA molecule allows this base to pair with either U, C or A in the third 3' position of a messenger codon. Similarly, with G or U in the 5' position in the anticodon, two codons may be recognized by a single aminoacyl-tRNA, whereas with C or A only a single codon is recognized. Since alternative base-pairing is only possible with the base in the third position of the codon, codons for the same amino acid which differ in either of the first two base positions, base-pair with different tRNAs. The only example of 'wobble' in the first base of a codon occurs with tRNA_i^Met , which recognizes both the initiator AUG or GUG codons (see later). Even so, separation of tRNAs by counter current-distribution, MAK columns, BD-cellulose columns and reversed phase chromatography has shown that there are more isoacceptor tRNAs than can be accounted for by the degeneracy of the genetic code. Similarly, isoacceptor tRNAs for amino acids exist in plants; for example, soyabean seedlings have at least six different tRNA^Leu species (Anderson & Cherry, 1969). Although few experiments have been carried out, it has been found in every case investigated that plants use the same codon in amino acid assignments as those of E. coli. However, plants may not use all of the possible codons for a particular amino acid, since some isoacceptor tRNA species may be absent, or because of the 'wobble' effect; e.g. Caskey et al., (1968) have shown that the isoleucyl tRNA from guinea pig liver recognizes AUU, AUC and AUA codons, whereas the corresponding isoleucyl-tRNA from E. coli only recognizes two of these, AUU and AUC. This response to different codons in the two organisms is explained if the liver tRNA had 5' inosine in the anticodon, while E. coli tRNA had guanine.

The whole question of the number and the activity of tRNAs in different tissues of plants is complicated by the fact that, (a) it is not certain whether the methods for separating tRNAs are completely effective; (b) it is known that during isolation and purification, partial modification of tRNA molecules can occur, which may affect their charging ability with amino acids or their ability to transfer amino acids to proteins, or both (see Sueoka & Kano-Sueoka, 1970); (c) the relative importance of the three protein synthesizing systems, cytoplasmic, chloroplast and mitochondrial, each with its associated tRNAs and synthetases may differ in different tissues. Nevertheless, different complements of isoacceptor tRNAs may occur in different tissues and changes in pattern have been demonstrated during development (Bick et al., 1970; Littauer & Inouye, 1973), and it has been suggested that isoacceptor tRNAs are involved in the control of protein synthesis. Garel et al., (1973) have shown that the complement of tRNAs synthesized in the silk gland is related to the mRNAs being translated and they have suggested defining this phenomenon as 'modulated tRNA biosynthesis'.

10.3.1.2—
Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases have been isolated and purified from a variety of plants (see Boulter 1970; Boulter et al., 1972; Zalik & Jones, 1973), and although a considerable amount of work has been done on their properties, several problems remain. For example, the question of the detailed substrate specificity of different aminoacyl-tRNA synthetases is still under investigation. Since activation is a two-step process, both amino acid and tRNA specificity have to be considered. The synthetase is normally absolutely specific towards the tRNA; few mismatches have been reported in homologous systems (Arca et al., 1967, 1968). However, a synthetase specific for one amino acid can, in some instances, activate to a lesser extent another amino acid. Even so, synthetases are remarkably amino acid specific. A second amino acid attached to a tRNA molecule normally specific for another amino acid, will be located in the polypeptide chain according to the tRNA anticodon, i.e. where the first amino acid would have been placed. With regard to amino acids not normally found in the cell, specificity is not always so pronounced, e.g. azetidine-2-carboxylic acid is activated and transferred to tRNA^Pro by the proline enzyme of mung bean (Peterson & Fowden, 1965), whereas Polygonatum multiflorum, which contains a high level of this amino acid in nature, contains a prolyl-tRNA synthetase which will not activate it. In Mimosa and Leucaena, where mimosine, an analogue of phenylalanine, occurs naturally and where it is non-toxic, the phenylalanyl-tRNA synthetase discriminates against mimosine. In several other species where mimosine is toxic, it is activated by the phenylalanine-tRNA synthetase but the enzyme does not attach it to tRNA^Phe (Smith & Fowden, 1968).

It is not clear if there is one or more than one synthetase whenever there are

a number of isoacceptor tRNAs. In soybean seedlings where there are at least three different leucyl aminoacyl-tRNA synthetases (Kanabus & Cherry, 1971) which have different specificities towards the six leucine isoacceptor tRNAs, it would appear that there is more than one cytoplasmic leucyl-tRNA synthetase. However, in some instances, a single aminoacyl-tRNA synthetase can recognize different isoacceptor tRNAs. When both cytoplasmic and chloroplast tRNA species are present, there are probably at least two sets of aminoacyl-tRNA synthetases. In light-grown Euglena for example (Reger et al., 1970; Krauspe & Parthier, 1973), there are two aminoacyl-tRNA synthetases for each of phenylalanine and leucine, whereas dark-grown Euglena, which is without chloroplasts, contains only one; there are various reports of chloroplast aminoacyl-tRNA synthetases which only acylate chloroplast tRNAs and not their cytoplasmic counterparts and vice versa. However, in heterologous systems such as chloroplast tRNA plus cytoplasmic activating enzymes, specificity may range from none to complete (Burkard et al., 1970; Boulter et al., 1972), although the biological significance of these findings is not clear.

There is evidence that, as well as the tRNAs, the complement of aminoacyltRNA synthetases may change during the life cycle of plants (Bick & Strehler, 1971).