Ala-tRNA

FIGURE 27-18 Structural elements of tRNAAla that are required for recognition by Ala-tRNA synthetase. 

Several aaRSs possess amino acid editing domains, which hydrolytically clear mistakes in cis (3) (Table 1). Interestingly however, some archaeal and bacterial synthetases rely on paralogs of editing domains that hydrolyze mischarged products in tram (10, 11). These products include the YbaK and ProX (or PrdX), which edit Ala-tRNA , and AlaX that hydrolyzes mischarged Ser-tRNA and Gly-tRNA . 

In an experiment, it was found that Cys-tRNA can be converted to Ala-tRNA -T and used in an in vitro system that is capable of synthesizing proteins. 

Whenever tested, binding of labels to the ribosome has been shown to be stimulated severalfold by the appropriate or by initiation factors. -" Based on reactivity with puromycin, all pep-tidyl-tRNA probes tested were considered to occupy predominantly the ribosomal donor site. This conclusion was supported by the observed ability of several Phe-tRNA derived probes to form a peptide bond with added Phe-tRNA as acceptor. - - Similarly, modified Met-tRNA bound with f2-RNA has been shown to interact with Ala-tRNA to form the Met-Ala initiation sequence typical of the phage coat protein. ... 

Yeast Ala tRNA Gene," in Proceedings of the Seventh International Congress of Biochemistry, Tokyo, August 19-25, 1967, p. 17. 

When a wrong amino acid is placed on the transfer RNA, for example when Cys-tRNA is chemically reduced to Ala-tRNA > t. the synthetase (Cys) becomes unable to remove the wrong amino acid (Ala), finally resulting in the synthesis of a wrong protein. A correct insertion is thus entirely dependent upon the conformable sites involved in the mutual recognition of the transfer RNA, and the aminoacyl-RNA synthetase. 

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

A tRNA molecule that is supposed to carry cysteine (tRNAcys) is mischarged, so that it actually carries alanine (ala-tRNAcys). What will be the fate of this alanine residue during protein synthesis ... 

These effects are nicely illustrated by changes at position 51.20,21 The tyrosyl-tRNA synthetase from B. stearothermophilus has Thr at this position. The tyrosyl-tRNA synthetase from Bacillus caldotenax differs by just four amino acid residues from that from B. stearothermophilus, but one of the changes is an Ala at position 51 23 The enzyme from E. coli is only 50% identical in sequence but has Pro at... 

Ledoux, S., Olejniczak, M., and Uhlenbeck, O. C. (2009). A sequence element that tunes Escherichia coli tRNA(Ala) (GGC) to ensure accurate decoding. Nat. Struct. Mol. Biol. 16, 359-364. 

Does this mischarged tRNA recognize the codon for cysteine or for alanine The answer came when the tRNA was added to a cell-free protein-synthesizing system. The template was a random copolymer of U and G in the ratio of 5 1, which normally incorporates cysteine (encoded by UGU) but not alanine (encoded by GCN). However, alanine was incorporated into a polypeptide when Ala-tHNA ys was added to the incubation mixture. The same result was obtained when mRNA for hemoglobin served as the template and [i CJalanyl-tRNACys was used as the mischarged aminoacyl-tRNA. The only radioactive tryptic peptide produced was one that normally contained cysteine but not alanine. Thus, the amino acid in aminoacyl-tRNA does not play a role in selecting a codon. 

---