Amino acids aminoacyl-tRNA synthetases

Process of charging tRNA with amino acids aminoacyl-tRNA synthetase enzymes... 

Esters of the substrate amino acids with alcohol residues of appreciable size are accepted by gramicidin S-synthetase and catalyze biosynthesis of gramicidin S (5). The activation mechanism for these compounds still needs to be elucidated. It is interesting that these findings are in contrast to the features observed for the ami noacyl adenylate activation of amino acids by tRNA synthetases in protein biosynthesis which are inhibited by substrate esters. From these results we infer that as in the tRNA synthetases the a-amino group of the substrate amino acids of gramicidin S synthetase is essential for their binding to the aminoacyl adenylate activation sites of the... 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific amino acid. 

Figure 38-1. Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme aminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction involves the formation of an AMP-amino acid-enzyme complex. This activated amino acid is next transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the latter can be reutilized. The charging reactions have an error rate of less than 10" and so are extremely accurate.

In 1994, a conference with the title Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code was held in Berkeley, California its patron was the Institute of Advanced Studies in Biology. The conference dealt with the development of the synthetases and that of the genetic code (see Sect. 8.2), i.e., the assignment of the various amino acids to the corresponding base triplets of the nucleic acids. 

Fig. 5.1 Simplified model representation of the activation of an amino acid (ASY) at an amino acid-activating enzyme (i.e., an amino acid-specific aminoacyl-tRNA synthetase)...

It was earlier considered that all the amino acid-activating synthetases were derived from a single primeval synthetase , so that all synthetases would have similar structures. Surprisingly, however, this is not the case. When the primary sequences, and in part the secondary and tertiary structures, of all the synthetases had been determined, clear differences in their construction became obvious. The aminoacyl-tRNA synthetases consist either of one single polypeptide chain (a) or of two or four identical polypeptides (ot2 or 04). In addition, there are heterogeneously constructed species with two sets of two identical polypeptide chains (OC2P2). This nomenclature indicates that, for each synthetase, a or P refers to a primary structure. The number of amino acids can vary from 334 to more than 1,000. 

Cech s group was the first to have success in this direction (Piccirilli, 1992). Using a genetically modified Tetrahymena ribozyme, they were able to hydrolyse an ester bond between the amino acid A-formylmethionine and the corresponding tRNAf Met. The reaction was, however, very slow, only about 5 to 15 times faster than the uncatalysed reaction. The authors ventured to suggest that these ribozymes could have functioned as the first aminoacyl tRNA synthetases. 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

An important factor in the evolution of the genetic code is certainly provided by the aminoacyl-tRNA synthetases (see Sect. 5.3.2). It is clear that the two synthetase classes are not randomly distributed across the matrix of the amino acid assignment of the genetic code. For example, with one exception, all XUX codons code for class 1 synthetases, while all XCX codons code for class 2 aminoacyl-tRNA synthetases. A possible explanation could be that the synthetases and the genetic code evolved simultaneously. However, it is more likely that these enzymes evolved when the genetic code had already been established (Wetzel, 1995). 

Protocol The test for the identification of aminoacyl-tRNA synthetase inhibitors requires the availability of precharged fMet-tRNA, Phe-tRNA, Thr-tRNA, and Ile-tRNA, which correspond to the amino acids present in the 027 peptide. The preparation of fMet-tRNA is described in the accompanying chapter by Milon et al., (2007), while the preparation of the other aminoacyl-tRNAs has been described previously. 

The tRNA synthetases may provide a check to make sure that the correct amino acid has been attached to the correct tRNA. If an incorrect amino acid is attached to the tRNA, it will be incorporated into the protein at the position specified by the identity of the tRNA. At least some of the aminoacyl tRNA synthetases have a proofreading function that hydrolyzes any incorrect aminoacyl tRNAs (for example, a Val residue attached to an lie tRNA). 

Aside from these relatively direct applications of site-directed mutagenesis, combination of recombinant DNA techniques with other experimental strategies will no doubt prove to be of increasing importance. If the gene of interest can be expressed with sufficient efficiency in auxotrophs, then proteins in which selected amino acids are isotopically enriched [12] may be produced to increase the sensitivity and selectivity of magnetic resonance techniques. Alternatively, amino acid analogues that are recognized as substrates by aminoacyl tRNA synthetases may be incorporated randomly in place of the true substrate amino... 

For most amino acids, the ester linkage between the ct-COOH group of the amino acid and the 3 -terminal adenosine of a cognate tRNA is formed in a two-step mechanism catalyzed by an aminoacyl-tRNA synthetase (aaRS). ° In this so-called direct pathway, the aaRS first catalyzes the reaction of the amino acid with adenosine triphosphate (ATP), yielding the enzyme-bound high-energy intermediate aa AMP and PPi in the second step, this aaRS-bound intermediate reacts with tRNA to yield aa-tRNA and AMP (Figure 1). 

Aminoacyl-tRNA synthetases charge the appropriate tRNA with the correct amino acid, which is important in maintaining the fidelity of protein translation. To genetically encode an unnatural amino acid, the substrate specificity of the orthogonal synthetase needs to be altered to charge the orthogonal tRNA with only the desired unnatural amino acid and none of the common 20 amino acids. A general scheme was developed for... 

Figure 3 A general positive and negative selection strategy for evolving aminoacyl-tRNA synthetase variants specific for an unnatural amino acid.

Each type of amino acid is activated by a different aminoacyl tRNA synthetase. 

The aminoacyl tRNA synthetase transfers the activated amino acid to the 3 end of the correct tRNA... 

Amino acid activation Aminoacyl-tRNA synthetase two high-energy bonds (ATP) to link amino acid to tRNA j ... 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

There is a family of enzymes that catalyze the attachment of amino acids to then-cognate tRNAs, aminoacyl-tRNA synthetases. There is one or more of these enzymes for each of the 20 amino acids that occur commonly in proteins. Each of these enzymes recognizes (a) a specific amino acid and (b) its cognate tRNA. Imagine a soup of 20 amino acids and 20 tRNAs, one for each amino acid. For example, the aminoacyl-tRNA synthetase for, saline would specifically pick valine out of the soup and catalyze its attachment to the tRNA for valine, tRNA . Simply, we can write the product of the reaction as val-tRNA . This is a lovely example of the role of molecular recognition in a critical life process. 

All tRNA molecules have the sequence -CCA at the 3 end. This three base sequence is termed the acceptor stem. The aminoacyl-tRNA synthetases catalyze the formation of an ester between the carboxyl group of the amino acid and the 3 -OH of the ribose of the terminal adenosine moiety ... 

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