Aminoacylation of tRNAs

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. 

Robert Chenevert is Professor of Organic Chemistry at Universite Laval, Quebec, Canada. He studied chemistry (B.Sc. and M.Sc.) at the Universite de Montreal. After receiving his Ph.D. in organic chemistry in 1975 at the Universite de Sherbrooke under the supervision of Professor Pierre Deslongchamps, he spent a postdoctoral year at Harvard (R. B. Woodward s group). His main research interest is the application of biocatalysts in asymmetric synthesis. He is also interested in the design of inhibitors of enzymes involved in the aminoacylation of tRNA (aminoacyl-tRNA synthetases and aminoacyl-tRNA amidotransferases). 

MECHANISM FIGURE 27-14 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, (2a) the aminoacyl group is transferred initially to the 2 -hydroxyl group of the 3 -terminal A residue, then (3a) to the 3 -hydroxyl group by a transesterification reaction. For class II enzymes, ( the... 

When IRS, isoleucine, tRNA, and [y-32P]ATP (labeled in the terminal phosphate) are mixed in the pulsed quenched-flow apparatus (Figure 7.5), there is a burst of release of labeled pyrophosphate before the steady state rate of aminoacylation of tRNA is reached. This means either that the aminoacyl adenylate is formed before the aminoacylation of tRNA, thus proving the... 

By virtue of their long evolutionary history, as weU as of their capacity to bind RNA, ATP, and other small molecules such as amino acids, the aaRSs have been recruited to carry out many diverse alternative functions in cells (4) (Fig. 4a). One such function includes capitalizing on its aminoacylation function to proofread tRNA processing and maturation in the nucleus that occurs before tRNA export to the cytoplasm for protein synthesis. In addition, aminoacylation of tRNA-like structures, such as the 3 -end of viral genomes in plants and tmRNA in Escherichia coli, is important for viral replication and for ribosome recycling, respectively. 

Robertson SA, Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG. The use of 5 -phospho-2 deoxyrihocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA. Nucleic Acids Res. 1989 17 9649-9660. 

This enzyme catalyses the aminoacylation of tRNA in a two-step reaction... 

R. Lund and J. E. Dahlberg Proofreading and aminoacylation of tRNAs before export from the nucleus. Science 282,2082 (1998). 

There are 20 different aminoacyl-tRNA synthetases, the enzymes that covalently link the amino acid to the correct tRNA. We will denote the tRNA species as tRNAAA and the charged tRNA as AA-tRNAAA. Charging must be a very accurate system because the measured error rate (when a tRNA is charged with the wrong amino acid) is less than 10-4. Aminoacyl-tRNA synthetases are bifunctional in that they are capable of distinguishing between all 20 amino acids, and the full complement of cellular tRNAs. In E. coli, 20 aminoacyl-tRNA synthetases have been identified (one for each amino acid), indicating that related tRNAs, such as the six possible tRNALeu species, must be recognized by the same enzyme. A two-step reaction is required for the aminoacylation of tRNAs ... 

Aminoacylation of tRNA with Nonnatural Amino Acids... 

Fig. 5.1-2 Hecht method for chemical aminoacylation of tRNA with a nonnatural amino acid.

At this moment, aminoacylation of tRNA with a nonnatural amino acid is still a bottleneck step for nonnatural mutagenesis both in vitro and in vivo. Hecht method is versatile to almost any types of amino acids, but can be done only for isolated tRNAs in a test tube. Further, the aminoacylation step of pdCpA is sometimes tricky. For aminoacylation in a test tube, micelle-mediated method is easier than the Hecht method, at least for some types of amino acids. The ribozyme technique of Suga is applicable to a variety of p-substituted phenylalanines and to a wide variety of tRNAs. This is, at present, the simplest and most dependable method of aminoacylation for isolated tRNAs. It has not been, however, applied to in vivo systems and to large-sized amino acids. Our PNA-assisted aminoacylation method may also be applicable to a wide variety of amino acids and tRNAs. Since the PNA-assisted aminoacylation is tRNA selective, it works as a potential amino acid donor in living cells. The orthogonal tRNA/aaRS pairs reported by Schultz and by Yokoyama are effective in some nonnatural amino acids with small side groups, but they have not been applied to large-sized amino acids, so far. 

N. Hashimoto, K. Ninomiya, T. Endo, M. Sisido, Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation, Chem. Commun. 2005, 4321-4323. 

To illustrate the explicit role of water here we consider three biochemical processes (a) aminoacylation of tRNA in the presence of aminoacyl-tRNA synthetase, (b) translation in the ribosome, and (c) DNA replication. In the case... 

Variations in the level of aminoacylation of tRNA arising from the altered specificity of an aminoacyl-tRNA synthetase or from an amino acid depletion would be expected to exert a profound effect on the rate and level of protein synthesis. There are reports that qualitative changes in aminoacyl-tRNA synthetases may occur during embryo development [58] and in different tissues and organs [15] and that multiple synthetases may occur [59]. However, with the knowledge that mitochondria contain unique synthetases, a reexamination of these data in terms of the localization of the enzymes would help in clarification. 

Figure 57 Acyl transfer from ester 162 to alcohol 163 catalyzed by antibody 13D6.1 raised against hapten 161. (when T = thymidyl the reaction is a model for the aminoacylation of tRNA).

---