Aminoacyl-tRNA mischarged

There are two high-energy intermediates on the reaction pathway that could be edited by hydrolysis the enzyme-bound aminoacyl adenylate and the aminoacyl-tRNA. A mechanistic study must distinguish between the two. A pathway involving the mischarged tRNA involves the formation of a covalent intermediate—the aminoacylated tRNA—so the three rules of proof may be considered (Chapter 7, section Al). These criteria have been rigorously applied to the rejection of threo-... 

Aminoacyl-tRNA synthetases (aaRSs) compose a family of essential enzymes that attach amino acids covalently to tRNA molecules during protein synthesis. Some aaRSs possess a hydrolytic amino acid editing function to ensure the fidelity of protein synthesis. In addition, aminoacylation can occur by indirect pathways that rely on mischarged tRNA intermediates and enzymes other than aaRSs. Throughout evolution, structural and functional divergence of aaRSs has yielded diverse secondary roles. 

Threonyl-tRNA synthetase can be incubated with tUNA hr that has been covalently linked with serine (Ser-tRNAThr) the tRNA has been "mischarged." The reaction is immediate a rapid hydrolysis of the aminoacyl-tRNA forms serine and free tRNA. In contrast, incubation with correctly charged Thr-tRNAThr results in no reaction. Thus, threonyl-tRNA synthetase contains an additional functional site that hydrolyzes Ser-tRNA hr but not Thr-tHNA hr This editing site provides an opportunity for the synthetase to correct its mistakes and improve its fidelity to less than one mistake in 10. The results of structural and mutagenesis studies revealed that the editing site is more than 20 A from the activation site (Figure 29.9). This site readily accepts and cleaves Ser-tRNAThr but does not cleave Thr-tRNA hr The discrimination of serine from threonine is relatively easy because threonine contains an extra methyl group a site that conforms to the structure of serine will sterically exclude threonine. 

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. 

Figure 1 Aminoacylation reaction, (a) The overall aminoacylation reaction is performed in two steps by the aaRSs. Two modes of amino acid editing can hydrolyze the mischarged tRNA product (posttransfer editing) or misactivated aminoacyl adenylate intermediate (pretransfer editing), (b) The first step of the ATP-dependent aminoacylation reaction activates amino acid to generate an aminoacyl adenylate intermediate, (c) In the second step, the activated amino acid is transferred to the tRNA molecule and AMP Is released.

Site-specific insertion of novel amino acids in proteins originally was accomplished in vitro by generating mischarged suppressor tRNAs, which have anticodons that pair with amber stop codons. The tRNAs were aminoacylated chemically with natural and novel amino acids and were then incorporated at specific stop codons using in vitro translation systems. 

A third mechanism of synthesis, which was only recently recognized, appears to provide the sole source of asparagine for many bacteria. The asparagine-specific transfer RNA tRNA " is "mischarged" with aspartic acid to form Asp-tRNA ". This compound is then converted to the properly aminoacylated Asn-tRNA " by a glutamine-dependent amidotransferase. (The entire ATP-dependent sequence is shown in Eq. 

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