From the Skaggs Institute for Chemical Biology and Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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The fidelity of protein synthesis requires
efficient discrimination of amino acid substrates by aminoacyl-tRNA
synthetases. Accurate discrimination of the structurally similar amino
acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS)
results, in part, from a hydrolytic editing reaction, which prevents
misactivated valine from being stably joined to
tRNAIle. The editing reaction is dependent on the
presence of tRNAIle, which contains discrete D-loop
nucleotides that are necessary to promote editing of misactivated
valine. RNA minihelices comprised of just the acceptor-T Aminoacyl-tRNA synthetases establish the genetic code by attaching
amino acids to their cognate tRNAs (1-3). These reactions are
comprised of two steps. Initially the amino acid is condensed with ATP
to give an activated aminoacyl adenylate. Subsequently, the aminoacyl
moiety of this reactive intermediate is transesterified to the 3'
terminus of the tRNA. The fidelity of the genetic code depends upon the
precise molecular recognition of both the amino acid and tRNA
substrates by aminoacyl-tRNA synthetases. It has long been recognized
that the accurate transduction of molecular information is made
increasingly difficult when the molecular structures of two candidate
substrates are highly similar (4-7).
A prominent example of the need to discriminate between closely related
substrates is in the recognition of isoleucine over valine by
IleRS.1 Valine, which differs
from isoleucine by a single methylene unit, is activated by
Escherichia coli IleRS at a rate approximately 180-fold
slower than that of isoleucine (8). IleRS prevents this relatively high
error rate from being realized in protein synthesis through two editing
reactions that result in the net hydrolysis of misactivated valine (7,
9, 10). These reactions provide a second "sieve" (11) of
discrimination against valine and indeed occur at a second,
"editing" active site on IleRS (8, 12).
Editing of misactivated valine has a strict requirement for
tRNAIle (9, 13). In the absence of tRNAIle,
enzymatically generated Ile-AMP and Val-AMP remain sequestered in the
active site. Upon addition of tRNAIle to the
IleRS·Ile-AMP complex, the aminoacyl group is stably attached to
tRNAIle. In contrast, addition of tRNAIle to
the IleRS·Val-AMP complex results in immediate hydrolysis of the
valyl adenylate. This occurs partly through a pretransfer editing
reaction where misactivated Val-AMP is directly hydrolyzed. Alternatively, the valyl moiety of Val-AMP is transferred to
tRNAIle to make a transient Val-tRNAIle
intermediate that is rapidly hydrolyzed. The net result of either pathway is an abortive cycle of valine activation followed by tRNAIle-dependent hydrolysis that continues
until all available ATP is consumed.
In contrast to nucleotide determinants for charging that are located in
the anticodon loop and acceptor stem of tRNAIle (14),
nucleotides in the D-loop of tRNAIle trigger the editing
reaction (13). For example, replacement of G-16, D-20, and D-21 in the
D-loop of tRNAIle with their counterparts from
tRNAVal has little effect on aminoacylation. In contrast,
these substitutions abolish the editing response. Conversely, transfer
of the three D-loop nucleotides from tRNAIle into the
framework of a specially designed tRNAVal confers editing
activity to the chimerized tRNA. Thus, tRNA determinants for editing
and aminoacylation are discrete.
Similarly, a single (G56A) mutation in the active site (for
aminoacylation) of IleRS eliminates the discrimination between isoleucine and valine in amino acid activation (8). However, the G56A
mutant enzyme still discriminates between isoleucine and valine in the
post-transfer editing reaction. These and other mutational analyses,
along with chemical cross-linking data (12, 15), showed that the
editing and aminoacylation sites are physically distinct and
functionally independent. Specifically, the editing site is located
within a large insertion known as CP1 (connective polypeptide 1) (16).
This 277-amino acid insertion divides the characteristic class I
catalytic domain in half (17). A recently determined x-ray structure
places the two active sites about 25-Å apart (18).
A present working hypothesis is that specific nucleotides in the D-loop
of tRNAIle trigger the translocation of the valyl group
from the aminoacylation to the editing site. Here we tested whether the
presumptive translocation and editing response could be recreated by
dividing the critical domains of tRNAIle into two pieces.
One piece is an oligonucleotide substrate that recreates the acceptor
stem of the tRNA in the form of a minihelix. Previous work showed that
IleRS could charge minihelixIle with isoleucine (19, 20).
The other piece is an RNA hairpin ligand designed after the D-loop of
tRNAIle. Thus, we asked whether the two pieces in concert
could reproduce the editing reaction or whether continuity of the tRNA
structure was required.
If the editing response requires the full tRNAIle structure
and if the D-loop provides critical determinants only within the context of the full tRNA, then we imagined that
minihelixIle might be a good substrate for mischarging with
valine. In that event, we wondered whether mischarging depended on the
same nucleotide determinants as those required for correct
aminoacylation. This part of the analysis was motivated by the prospect
of discovering acceptor stem determinants that modulate discrimination
against valine.
Protein Expression and Purification--
Wild-type E. coli IleRS was overexpressed in the E. coli strain
MV1184 harboring the multicopy plasmid pKS21 (21). Protein purification
was essentially as described previously (22).
RNA Substrates--
RNA hairpins were chemically synthesized
using N-phenoxyacetyl-protected ribonucleoside
phosphoramidites (ChemGenes, Waltham, MA) on a Amersham Pharmacia
Biotech Gene Assembler Special (23). RNA concentrations were determined
using absorbance at 260 nm at room temperature. Extinction coefficients
were estimated using the Biopolymer Calculator available
online.2 Mature E. coli tRNA1Ile (GAU) was isolated
from E. coli strain MV1184 containing the plasmid pES300,
which allows for the lac-inducible overexpression of
tRNAIle (24, 25).
Editing Assays--
The RNA-dependent hydrolysis of
valyl adenylate was assayed by following the consumption of
[ Aminoacylation Assays--
The IleRS-catalyzed isoleucylation or
valylation of RNA substrates was followed using the trichloroacetic
acid precipitation method of Shepard and co-workers (22). Reactions
were carried out at room temperature in a solution containing 20 mM HEPES (pH 7.5), 150 mM NH4Cl, 20 mM MgCl2, 100 µM EDTA, 2 mM ATP, 10 nM inorganic pyrophosphatase, 5 µM [3H]amino acid (~20 mCi/µmol), and 5 µM IleRS. RNA minihelices were used at a concentration of
50-500 µM and tRNAIle at a concentration of
10-40 µM. No-RNA controls were used to correct for
background rates.
RNA-dependent Hydrolysis of Misactivated
Valine--
The L-shaped tRNAIle structure (Fig.
1, top right) is composed of
two helical domains (27-29). Coaxial stacking of the acceptor and
T
At least half of the tRNA synthetases charge minihelices based on the
acceptor-T
MinihelixIle (Fig. 1, bottom) contains
determinants for aminoacylation by IleRS (19, 20). Previously, this
domain of tRNAIle had not been investigated for its ability
to stimulate editing of misactivated valine. As can be seen in Fig.
2, the addition of tRNAIle to
a mixture of IleRS, ATP, and valine rapidly leads to the complete consumption of available ATP. In contrast, minihelixIle is
unable to stimulate the hydrolytic editing of misactivated valine, even
at the high concentration of 200 µM.
Next we tested whether the addition of an isolated D-stem/loop domain
of tRNAIle could induce editing. For this purpose, we
constructed a 9-base pair RNA hairpin that extends the D-stem by
pairing nucleotide G-26 and including 4 base pairs of the anticodon
stem (D-loopIle, Fig. 1, top left). This
D-stem/loop RNA hairpin did not induce editing (see Fig. 2). Finally,
we obtained no evidence for an editing response when
minihelixIle and D-loopIle were used in
combination (data not shown for clarity). These results suggest that
continuity of the tRNAIle structure is required for the
editing response.
Mischarging of a Minihelix Substrate--
Because
minihelixIle is capable of being aminoacylated with
isoleucine and yet is unable to induce editing, we tested its ability to be aminoacylated with valine. As shown in Fig.
3, minihelixIle is a
relatively robust substrate for mischarging with valine. This level of
mischarging was not observed with mature tRNAIle, as the
editing reaction prevents the stable attachment of valine to
tRNAIle. We tried to force misacylation of
tRNAIle by using concentrations (40 µM) well
above the Km (~5 µM, Ref. 14).
Still, no misacylation of the full tRNA could be detected. Thus,
although minihelixIle is significantly less active than
tRNAIle for charging with isoleucine, it is far more active
in mischarging with valine.
Mischarging of Minihelix Sequence Variants--
Despite the
reduced activity of minihelixIle for charging with
isoleucine, this aminoacylation is specific. For example, substitution of the A-73 discriminator with G results in a minihelix that is 8.4-fold less active in charging with isoleucine. A qualitatively similar effect is seen when the same substitution is made in the full
tRNA (14). Here we found that mischarging A73G minihelixIle
with valine showed the same rate reduction that was observed for
charging with isoleucine (Fig. 4). In
addition, we investigated a variant of minihelixIle
designated
A direct comparison of the initial rates of aminoacylation with both
isoleucine and valine reveals that isoleucine is only a 3-fold better
substrate under these conditions. This level of discrimination was
observed over a large concentration range (50-500 µM) of
minihelix substrate (data not shown), showing that the nature of the
amino acid does not modulate minihelix binding. Because the rates of
Ile-AMP and Val-AMP synthesis are greater than the rate of
aminoacylation of minihelix substrates, it is likely that the aminoacyl
adenylates (of both isoleucine and valine) accumulate in the active
site to similar levels. Under these circumstances, the rate of
aminoacyl-RNA formation should be most correlated to the transfer rate
of the aminoacyl group from the adenylate to the minihelix. The small
difference between the isoleucylation and valylation rate of
minihelixIle may represent a lack of discrimination toward
the amino acid side chain in the transfer step.
The ability of isoleucyl-tRNA synthetase to discriminate against
valine is enhanced in the presence of tRNAIle. In charging
of minihelix substrates, this discrimination is reduced to a mere
3-fold preference for isoleucine. This outcome is expected for
substrates that have significantly reduced editing activity and yet
have retained some charging activity. The mischarging of a minihelix
reinforces the evidence that the D-loop of tRNAIle is
indispensable for proper discrimination against valine (13). To some
extent this finding has been puzzling, as it is thought that IleRS
makes little contact with the D-loop of tRNAIle; for
example, none of the D-loop nucleotides critical for editing are
protected by bound IleRS in phosphate ethylation experiments (14).
Considering the available evidence, we propose that the effect of the
D-loop is derived from its presumed role in transducing conformational
information between the two domains of tRNAIle. Long range
information transfer through the tRNA structure is also suggested from
studies of the aminoacylation of tRNAIle by IleRS. For
example, mutations in the anticodon of tRNAIle (which
interacts directly with IleRS) do not affect binding but rather
diminish the kcat for charging. In a model for
aminoacylation of wild-type tRNAIle, binding of the correct
anticodon enables conformational changes that accurately orient the
acceptor terminus in the distant active site (14).
The charging of minihelices by IleRS provides a model aminoacylation
system where one or more of the discriminatory "sieves" has been
attenuated. It should be noted that the notion of double sieve discrimination is actually semantic in that editing by IleRS is
known to involve two editing reactions (i.e. pre- and
post-transfer). Considering the initial activation sieve, there are at
least three functional sieves. In theory, discrimination
against valine could involve numerous other sieves, each acting at a
unique kinetic step along the aminoacylation reaction coordinate. One
such step that could enhance discrimination against valine is the
transfer of the aminoacyl group from the activated adenylate to the
accepting 2'-OH of the RNA. However, our results suggest that this is
not the case.
In the aminoacylation of minihelices (where the aminoacylation rate is
likely to reflect the transfer rate) the preference for isoleucine is
quite small. Mutations in minihelix substrates that affect the
aminoacylation rate do so independently of the amino acid side chain.
At least for the Several considerations have led to the hypothesis that minihelices were
evolutionary precursors to modern tRNAs (37-40) and had an origin
distinct from that of the anticodon-containing domain (41). The
correlation of acceptor stem nucleotides in minihelices with the
charging of specific amino acids constitutes an operational RNA code
(that could have preceded the anticodon-dependent genetic code (42)) for amino acids (39). Therefore, it is interesting to wonder
whether the relaxed amino acid specificity in the aminoacylation of
minihelixIle reported here is indicative of more primitive
aminoacylation systems in general. Perhaps during the early development
of an operational RNA code aminoacylation systems with greater net
catalytic activity had a selective advantage over those with lower, yet more specific catalytic activity. Subsequently, as aminoacylation systems became more robust, specificity could have become a greater selective advantage. At this point, determinants for editing may have
been appended to the tRNA structure.
C helix of
tRNAIle are substrates for specific aminoacylation by
IleRS. These substrates lack the aforementioned D-loop nucleotides.
Because minihelices contain determinants for aminoacylation, we thought
that they might also play a role in editing that has not previously
been recognized. Here we show that, in contrast to tRNAIle,
minihelixIle is unable to trigger the hydrolysis of
misactivated valine and, in fact, is mischarged with valine. In
addition, mutations in minihelixIle that enhance or
suppress charging with isoleucine do the same with valine. Thus,
minihelixIle contains signals for charging (by IleRS) that
are independent of the amino acid and, by itself,
minihelixIle provides no determinants for editing. An RNA
hairpin that mimics the D-stem/loop of tRNAIle is also
unable to induce the hydrolysis of misactivated valine, both by itself
and in combination with minihelixIle. Thus, the native
tertiary fold of tRNAIle is required to promote efficient
editing. Considering that the minihelix is thought to be the more
ancestral part of the tRNA structure, these results are consistent with
the idea that, during the development of the genetic code, RNA
determinants for editing were added after the establishment of an
aminoacylation system.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]ATP using a protocol that has been described in
detail elsewhere (26). The reaction mixture contained 140 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
0.5 mM CaCl2, 2 mM
[
-32P]ATP (25 µCi/µmol), 1 mM valine,
75 nM inorganic pyrophosphatase, 5 µM IleRS,
and either 40 µM tRNAIle or 200 µM minihelixIle and/or D-loopIle.
Reactions with no RNA were used to control for a small background rate
of hydrolysis (typically about 1% of the
tRNAIle-dependent rate).
RESULTS
C stems gives rise to the hairpin minihelix domain as the upper arm
of the L-shape. The second domain is a "dumbbell" formed by the
anticodon and D-stem/loops. Specific hydrogen bonds between the T
C
and D-loops link the two helical domains at the corner of the L-shaped
tRNA. Interactions at the corner are crucial for establishing the
canonical tRNA fold, and thus the nucleotides that form these
interactions are well conserved among all tRNA species. This junction
forms an obvious connection through which signals might be transmitted
from one domain to the other. D-loop nucleotides previously identified
as essential for editing are marked with arrows in Fig. 1.
These nucleotides are not among those needed for the universal
connections between the two domains.
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Fig. 1.
RNA substrates for editing and/or
aminoacylation assays. The structure of tRNAIle
(top right) consists of two helical domains arranged in an
L-shape. The dotted lines denote tertiary
interactions (29). Arrows indicate nucleotides (G-16, D-20,
D-21) of tRNAIle that are important for the
RNA-dependent editing of misactivated valine.
D-loopIle (top left) is an RNA hairpin based on
the D-stem/loop of tRNAIle. MinihelixIle
(bottom) mimics the acceptor-T C helical domain of
tRNAIle. Two variants of the minihelix are shown:
1
minihelixIle is missing the 5'-terminal adenosine; A73G
minihelixIle has an A to G mutation at the discriminator
position 73.
C stem of their cognate tRNAs (30-33). Although certain
synthetases do not make any contacts with the anticodon (e.g. alanyl-tRNA synthetase (34) and seryl-tRNA synthetase (35)), many others interact with both the acceptor stem and anticodon.
In the latter cases, the enzymes can still aminoacylate their
associated minihelices, although the efficiency is generally severely
reduced relative to the full tRNA. (In the E. coli
isoleucine system studied here, the minihelix is
~106-fold less active than tRNAIle.)
Nevertheless, aminoacylation of minihelices generally retains the same
sequence specificity for acceptor stem nucleotides as seen in the
charging of tRNAs. For this reason, minihelices are thought to interact
with the active site in a way that closely parallels the full tRNA.
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Fig. 2.
RNA-dependent editing of
misactivated valine at pH 7.5, 25 °C. In the presence of
valine, ATP, and tRNAIle, IleRS rapidly hydrolyzes all the
ATP because of the editing of Val-AMP. Neither minihelixIle
nor D-loopIle induces editing.
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Fig. 3.
IleRS mischarges a minihelix substrate with
valine at pH 7.5, 25 °C. Under the same conditions IleRS is
unable to mischarge tRNAIle because of the editing
reactions.
1 minihelixIle, in which the 5'-terminal A-1
nucleotide has been deleted. Recently, we discovered that this deletion
enhances charging with isoleucine (20). Presumably, this disruption of
the first base pair increases the flexibility of the single-stranded 3'
terminus. (The 3'-end of bound tRNAGln in the co-crystal
with glutaminyl-tRNA synthetase is folded back into the active site
(36). Because glutaminyl-tRNA synthetase and IleRS are structurally
related, the enhanced flexibility of the
1 substrate is thought to
ease passage of the minihelix acceptor terminus into the transition
state for catalysis.) The enhancement observed in charging
1
minihelixIle with isoleucine was exactly paralleled in
misacylation with valine (Fig. 4). Thus, the determinants for charging
of minihelixIle are independent of which amino acid is used
as the substrate. These results rule out the possibility that the
acceptor stem plays a role in amino acid discrimination.
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Fig. 4.
Mutations in minihelixIle have
identical effects on aminoacylation with isoleucine and valine.
A, charging with isoleucine; B, charging with
valine.
DISCUSSION
1 minihelixIle mutation, we obtained
evidence that the mutation directly affects the transfer rate with
little or no effect on binding. Although the high Km
values (>200 µM) for these substrates make this
parameter difficult to measure precisely, approximate
Km values for minihelixIle and
1
minihelixIle are within 20% of each other (data not
shown). In contrast, the kcat value for
1
minihelixIle is more than 3-fold higher than that for
minihelixIle in charging with both isoleucine and valine.
These experiments offer further evidence that transfer of the aminoacyl
group from the activated adenylate to the RNA does not provide
significant discrimination against valine.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM15539.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Howard Hughes Medical Institute predoctoral fellow.
§ To whom correspondence should be addressed: The Scripps Research Inst., 10550 N. Torrey Pines Rd., Mail Code BCC-379, La Jolla, CA 92037. Tel.: 619-784-8970; Fax: 619-784-8990; E-mail: schimmel{at}scripps.edu.
2 A. Schepartz, web page address: http://paris.chem.yale.edu/extinct.html.
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ABBREVIATIONS |
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The abbreviations used are: IleRS, isoleucyl-tRNA synthetase; Ile-AMP, isoleucyl adenylate; Val-AMP, valyl adenylate.
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REFERENCES |
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