AminoacylTransfer RNA Synthetases Read the Genetic Code

III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.1. Protein Synthesis Requires the Translation of Nucleotide Sequences Into Amino Acid Sequences
Figure 29.6. Helix Stacking in tRNA. The four helices of the secondary structure of tRNA (see Figure 29.4) stack to
form an L-shaped structure.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
The linkage of an amino acid to a tRNA 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 (Figure 29.7).
29.2.1. Amino Acids Are First Activated by Adenylation
The activation reaction is catalyzed by specific aminoacyl-tRNA synthetases, which are also called activating enzymes.
The first step is the formation of an aminoacyl adenylate from an amino acid and ATP. This activated species is a mixed
anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP; hence, it is also
known as aminoacyl-AMP.
The next step is the transfer of the aminoacyl group of aminoacyl-AMP to a particular tRNA molecule to form
aminoacyl-tRNA.
The sum of these activation and transfer steps is
The ∆ G° of this reaction is close to 0, because the free energy of hydrolysis of the ester bond of aminoacyl-tRNA is
similar to that for the hydrolysis of ATP to AMP and PPi. As we have seen many times, the reaction is driven by the
hydrolysis of pyrophosphate. The sum of these three reactions is highly exergonic:
Thus, the equivalent of two molecules of ATP are consumed in the synthesis of each aminoacyl-tRNA. One of them is
consumed in forming the ester linkage of aminoacyl-tRNA, whereas the other is consumed in driving the reaction
forward.
The activation and transfer steps for a particular amino acid are catalyzed by the same aminoacyl-tRNA synthetase.
Indeed, the aminoacyl-AMP 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 (Section 22.2.2). The major
difference between these reactions is that the acceptor of the acyl group is CoA in fatty acid activation and tRNA in
amino acid activation. The energetics of these biosyntheses are very similar: both are made irreversible by the hydrolysis
of pyrophosphate.
29.2.2. Aminoacyl-tRNA Synthetases Have Highly Discriminating Amino Acid
Activation Sites
Each aminoacyl-tRNA synthetase is highly specific for a given amino acid. Indeed, a synthetase will incorporate the
incorrect amino acid only once in 104 or 105 catalytic reactions. How is this level of specificity achieved? 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-tRNA synthetase reveals how valine is avoided (Figure 29.8).
The enzyme contains a zinc ion, bound to the enzyme by two histidine residues and one cysteine residue. Like carbonic
anhydrase (Section 9.2.1), 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 tRNAThr). Note that the carboxylate group of the amino acid is available
to attack the α-phosphate group of ATP 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 threonyltRNA synthetase.
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. Indeed, with only this mechanism available, threonyl-tRNA synthetase does mistakenly couple
serine to threonyl-tRNA at a rate 10-2 to 10-3 times that for threonine. As noted in Section 29.1.1, this error rate is likely
to lead to many translation errors. How is a higher level of specificity achieved?
29.2.3. Proofreading by Aminoacyl-tRNA Synthetases Increases the Fidelity of Protein
Synthesis
Threonyl-tRNA synthetase can be incubated with tRNAThr 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-tRNAThr but not Thr-tRNAThr. This editing site
provides an opportunity for the synthetase to correct its mistakes and improve its fidelity to less than one mistake in 104.
The results of structural and mutagenesis studies revealed that the editing site is more than 20 Å from the activation site
(Figure 29.9). This site readily accepts and cleaves Ser-tRNAThr but does not cleave Thr-tRNAThr. 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.
Most aminoacyl-tRNA 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-tRNA synthetase and its substrate reveals that the aminoacylated-CCA
can swing out of the activation site and into the editing site (Figure 29.10). 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 DNA polymerase (Section 27.2.4). In both cases,
editing without dissociation significantly improves fidelity with only modest costs in time and energy.
A few synthetases achieve high accuracy without editing. For example, tyrosyl-tRNA synthetase has no difficulty
discriminating between tyrosine and phenylalanine; the hydroxyl group on the tyrosine ring enables tyrosine to bind to
the enzyme 104 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.
29.2.4. Synthetases Recognize the Anticodon Loops and Acceptor Stems of Transfer
RNA Molecules
How do synthetases choose their tRNA 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. In a sense, aminoacyltRNA 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.
A priori, the anticodon of tRNA 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 tRNAThr (Figure 29.11). 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 G and U take part appear to be more important because the C 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 tRNAMet. Changing the anticodon sequence of this tRNA from CAU to GGU allows tRNAMet to be
aminoacylated by threonyl-tRNA synthetase nearly as well as tRNAThr, despite considerable differences in sequence
elsewhere in the structure.
The structure of another complex between a tRNA and an aminoacyl-tRNA synthetase, that of glutaminyl-tRNA
synthetase, again reveals extensive interactions with both the anticodon loop and the acceptor stem (Figure 29.12). In
addition, contacts are made near the "elbow" of the tRNA molecule, particularly with the base pair formed by G in
position 10 and C 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 tRNACys differs from tRNAAla 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 · U base
pair, tRNACys is recognized by alanyl-tRNA synthetase as though it were tRNAAla. 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 (Figure 29.13). Thus, specific aminoacylation is possible
for some synthetases even if the anticodon loop is completely lacking.
29.2.5. Aminoacyl-tRNA Synthetases Can Be Divided into Two Classes
Structural Insights, Aminoacyl-tRNA Synthetases. The first parts of the
tutorial focus on the structural differences that distinguish class I and class II
aminoacyl-tRNA 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-tRNA 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 (Table 29.2).
Glutaminyl-tRNA synthetase is a representative of class I. The activation domain for class I has a Rossmann fold
(Section 16.1.10). Threonyl-tRNA synthetase (see Figure 29.11) 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 (Figure 29.14). The CCA arm of tRNA adopts different conformations to accommodate these interactions; the
arm is in the helical conformation observed for free tRNA (see Figures 29.5 and 29.6) for class II enzymes and in a
hairpin conformation for class I enzymes. These two classes also differ in other ways.
1. Class I enzymes acylate the 2 -hydroxyl group of the terminal adenosine of tRNA, whereas class II enzymes (except
the enzyme for Phe-tRNA) acyl-ate the 3 -hydroxyl group.
2. These two classes bind ATP in different conformations.
3. Most class I enzymes are monomeric, whereas most class II enzymes are dimeric.
Why did two distinct classes of aminoacyl-tRNA 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.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.7. Aminoacyl-tRNA. Amino acids are coupled to tRNAs through ester linkages to either the 2 - or the 3 hydroxyl group of the 3 -adenosine residue. A linkage to the 3 -hydroxyl group is shown.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.8. 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 dimeric enzyme is shown in this and subsequent figures.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.9. Editing Site. The results of mutagenesis studies revealed the position of the editing site (shown in green) in
threonyl-tRNA synthetase.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.10. 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.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.11. Threonyl-tRNA Synthetase Complex. The structure of the complex between threonyl-tRNA synthetase
and tRNAThr reveals that the synthetase binds to both the acceptor stem and the anticodon loop.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.12. Glutaminyl-tRNA Synthetase Complex. The structure of this complex reveals that the synthetase
interacts with base pair G10:C25 in addition to the acceptor step and anticodon loop.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Figure 29.13. Microhelix Recognized by Alanyl-tRNA Synthetase. A stem-loop containing just 24 nucleotides
corresponding to the acceptor stem is aminoacylated by alanyl-tRNA synthetase.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.2. Aminoacyl-Transfer RNA Synthetases Read the Genetic Code
Table 29.2. Classification and subunit structure of aminoacyl-tRNA synthetases in E. coli
Class I
Class II
Arg (α) Ala (α 4)
Cys (α) Asn (α 2)
Gln (α) Asp (α 2)
Glu (α) Gly (α 2 β 2)