The linkage of an amino acid to a is crucial for two reasons. First, the attachment of a given amino acid to a particular tRNA establishes the genetic code. When an amino acid has been linked to a tRNA, it will be incorporated into a growing polypeptide chain at a position dictated by the anticodon of the tRNA. Second, the formation of a peptide bond between free amino acids is not thermodynamically favorable. The amino acid must first be activated for protein synthesis to proceed. The activated intermediates in protein synthesis are amino acid esters, in which the carboxyl group of an amino acid is linked to either the 2′- or the 3′-hydroxyl group of the ribose unit at the 3′ end of tRNA.
An amino acid ester of tRNA is called an aminoacyl-tRNA or sometimes a charged tRNA (). The activation and transfer steps for a particular amino acid are catalyzed by the same aminoacyl- synthetase. Indeed, the aminoacyl- intermediate does not dissociate from the synthetase. Rather, it is tightly bound to the active site of the enzyme by noncovalent interactions. Aminoacyl-AMP is normally a transient intermediate in the synthesis of aminoacyl-tRNA, but it is relatively stable and readily isolated if tRNA is absent from the reaction mixture. We have already encountered an acyl adenylate intermediate in fatty acid activation ().
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The major difference between these reactions is that the acceptor of the acyl group is in fatty acid activation and in amino acid activation. The energetics of these biosyntheses are very similar: both are made irreversible by the hydrolysis of pyrophosphate.
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Aminoacyl-tRNA Synthetases Have Highly Discriminating Amino Acid Activation Sites Each aminoacyl- synthetase is highly specific for a given amino acid. Indeed, a synthetase will incorporate the incorrect amino acid only once in 10 4 or 10 5 catalytic reactions. How is this level of specificity achieved?
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Each aminoacyl-tRNA synthetase takes advantage of the properties of its amino acid substrate. Let us consider the challenge faced by threonyl-tRNA synthetase. Threonine is particularly similar to two other amino acids—namely, valine and serine. Valine has almost exactly the same shape as threonine, except that it has a methyl group in place of a hydroxyl group. Like threonine, serine has a hydroxyl group but lacks the methyl group. How can the threonyl-tRNA synthetase avoid coupling these incorrect amino acids to threonyl-tRNA? The structure of the amino acid-binding site of threonyl- synthetase reveals how valine is avoided ().
The enzyme contains a zinc ion, bound to the enzyme by two histidine residues and one cysteine residue. Like carbonic anhydrase (), the remaining coordination sites are available for substrate binding. Threonine coordinates to the zinc ion through its amino group and its side-chain hydroxyl group.
The side-chain hydroxyl group is further recognized by an aspartate residue that hydrogen bonds to it. The methyl group present in valine in place of this hydroxyl group cannot participate in these interactions; it is excluded from this active site and, hence, does not become adenylated and transferred to threonyl-tRNA (abbreviated tRNA ). Note that the carboxylate group of the amino acid is available to attack the α-phosphate group of to form the aminoacyl adenylate. Other aminoacyl-tRNA synthetases have different strategies for recognizing their cognate amino acids; the use of a zinc ion appears to be unique to threonyl-tRNA synthetase.
Structure of Threonyl-tRNA Synthetase. The structure of a large fragment of threonyl-tRNA synthetase reveals that the amino acid-binding site includes a zinc ion that coordinates threonine through its amino and hydroxyl groups. Only one subunit of the The zinc site is less well suited to discrimination against serine because this amino acid does have a hydroxyl group that can bind to the zinc.
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Indeed, with only this mechanism available, threonyl- synthetase does mistakenly couple serine to threonyl-tRNA at a rate 10 -2 to 10 -3 times that for threonine. As noted in, this error rate is likely to lead to many translation errors. How is a higher level of specificity achieved?
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Editing Site. The results of mutagenesis studies revealed the position of the editing site (shown in green) in threonyl-tRNA synthetase. Most aminoacyl- synthetases contain editing sites in addition to acylation sites. These complementary pairs of sites function as a double sieve to ensure very high fidelity. In general, the acylation site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller than the correct one.
The structure of the complex between threonyl- synthetase and its substrate reveals that the aminoacylated-CCA can swing out of the activation site and into the editing site (). Thus, the aminoacyl-tRNA can be edited without dissociating from the synthetase. This proofreading, which depends on the conformational flexibility of a short stretch of polynucleotide sequence, is entirely analogous to that of polymerase (). In both cases, editing without dissociation significantly improves fidelity with only modest costs in time and energy. Editing of Aminoacyl-tRNA.
The flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site. If the amino acid fits well into the editing site, the amino acid is removed by hydrolysis. Few synthetases achieve high accuracy without editing. For example, tyrosyl- synthetase has no difficulty discriminating between tyrosine and phenylalanine; the hydroxyl group on the tyrosine ring enables tyrosine to bind to the enzyme 10 4 times as strongly as phenylalanine. Proof-reading has been selected in evolution only when fidelity must be enhanced beyond what can be obtained through an initial binding interaction. Synthetases Recognize the Anticodon Loops and Acceptor Stems of Transfer RNA Molecules How do synthetases choose their partners? This enormously important step is the point at which “translation” takes place—at which the correlation between the amino acid and the nucleic acid worlds is made.
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In a sense, aminoacyl-tRNA synthetases are the only molecules in biology that “know” the genetic code. Their precise recognition of tRNAs is as important for high-fidelity protein synthesis as is the accurate selection of amino acids. Priori, the anticodon of would seem to be a good identifier because each type of tRNA has a different one. Indeed, some synthetases recognize their tRNA partners primarily on the basis of their anticodons, although they may also recognize other aspects of tRNA structure. The most direct evidence comes from the results of crystallographic studies of complexes formed between synthetases and their cognate tRNAs.
Consider, for example, the structure of the complex between threonyl-tRNA synthetase and tRNA (). As expected, the CCA arm extends into the zinc-containing activation site, where it is well positioned to accept threonine from threonyl adenylate.
The enzyme interacts extensively not only with the acceptor stem of the tRNA, but also with the anticodon loop. The interactions with the anticodon loop are particularly revealing. The bases within the sequence CGU of the anticodon each participate in hydrogen bonds with the enzyme; those in which and take part appear to be more important because the can be replaced by G or U with no loss of acylation efficiency. The importance of the anticodon bases is further underscored by studies of tRNA. Changing the anticodon sequence of this tRNA from CAU to GGU allows tRNA Met to be aminoacylated by threonyl-tRNA synthetase nearly as well as tRNA Thr, despite considerable differences in sequence elsewhere in the structure.
Threonyl-tRNA Synthetase Complex. The structure of the complex between threonyl-tRNA synthetase and tRNA Thr reveals that the synthetase binds to both the acceptor stem and the anticodon loop. The structure of another complex between a and an aminoacyl-tRNA synthetase, that of glutaminyl-tRNA synthetase, again reveals extensive interactions with both the anticodon loop and the acceptor stem (). In addition, contacts are made near the “elbow” of the tRNA molecule, particularly with the base pair formed by in position 10 and in position 25 (denoted position 10:25). Reversal of this base pair from G C to C G results in a fourfold decrease in the rate of aminoacylation as well as a fourfold increase in the K M value for glutamine. The results of mutagenesis studies supply further evidence regarding tRNA specificity, even for aminoacyl-tRNA synthetases for which structures have not yet been determined. For example, E.
Coli tRNA differs from tRNA at 40 positions and contains a C G base pair at the 3:70 position. When this C G base pair is changed to the non-Watson-Crick G base pair, tRNA Cys is recognized by alanyl-tRNA synthetase as though it were tRNA Ala. This finding raised the question whether a fragment of tRNA suffices for aminoacylation by alanyl-tRNA synthetase. Indeed, a “microhelix” containing just 24 of the 76 nucleotides of the native tRNA is specifically aminoacylated by the alanyl-tRNA synthetase.
This microhelix contains only the acceptor stem and a hairpin loop (). Thus, specific aminoacylation is possible for some synthetases even if the anticodon loop is completely lacking. Structural Insights, Aminoacyl-tRNA Synthetases The first parts of the tutorial focus on the structural differences that distinguish class I and class II aminoacyl- synthetases. The final section of the tutorial looks at the editing process that most tRNA synthetases use to correct tRNA acylation errors. At least one aminoacyl- synthetase exists for each amino acid.
The diverse sizes, subunit composition, and sequences of these enzymes were bewildering for many years. Could it be that essentially all synthetases evolved independently? The determination of the three-dimensional structures of several synthetases followed by more-refined sequence comparisons revealed that different synthetases are, in fact, related. Specifically, synthetases fall into two classes, termed class I and class II, each of which includes enzymes specific for 10 of the 20 amino acids (). Glutaminyl-tRNA synthetase is a representative of class I. The activation domain for class I has a Rossmann fold (). Threonyl-tRNA synthetase (see ) is a representative of class II.
The activation domain for class II consists largely of β strands. Intriguingly, synthetases from the two classes bind to different faces of the tRNA molecule (). The CCA arm of tRNA adopts different conformations to accommodate these interactions; the arm is in the helical conformation observed for free tRNA (see and ) for class II enzymes and in a hairpin conformation for class I enzymes. These two classes also differ in other ways.
Classes of Aminoacyl-tRNA Synthetases. Class I and class II synthetases recognize different faces of the tRNA molecule.
The CCA arm of tRNA adopts different conformations in complexes with the two classes of synthetase. Class I enzymes acylate the 2′-hydroxyl group of the terminal adenosine of, whereas class II enzymes (except the enzyme for -tRNA) acyl-ate the 3′-hydroxyl group. These two classes bind in different conformations. Most class I enzymes are monomeric, whereas most class II enzymes are dimeric. Why did two distinct classes of aminoacyl- synthetases evolve? The observation that the two classes bind to distinct faces of tRNA suggests at least two possibilities. First, recognition sites on both faces of tRNA may have been required to allow the recognition of 20 different tRNAs.
Second, it appears possible that, in some cases, a class I enzyme and a class II enzyme can bind to a tRNA molecule simultaneously without colliding with each other. In this way, enzymes from the two classes could work together to modify specific tRNA molecules.