Magnesium Ion-mediated Binding to tRNA by an Amino-terminal
Peptide of a Class II tRNA Synthetase*
Rasha
Hammamieh and
David C. H.
Yang
From the Department of Chemistry, Georgetown University,
Washington, DC 20057
Received for publication, August 18, 2000, and in revised form, September 7, 2000
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ABSTRACT |
Aspartyl-tRNA synthetase is a class II tRNA
synthetase and occurs in a multisynthetase complex in mammalian cells.
Human Asp-tRNA synthetase contains a short 32-residue amino-terminal
extension that can control the release of charged tRNA and its direct
transfer to elongation factor 1
; however, whether the extension
binds to tRNA directly or interacts with the synthetase active site is
not known. Full-length human AspRS, but not amino-terminal 32 residue-deleted, fully active AspRS, was found to bind to noncognate tRNAfMet in the presence of Mg2+.
Synthetic amino-terminal peptides bound similarly to
tRNAfMet, whereas little or no binding of polynucleotides,
poly(dA-dT), or polyphosphate to the peptides was found. The apparent
binding constants to tRNA by the peptide increased with increasing
concentrations of Mg2+, suggesting Mg2+
mediates the binding as a new mode of RNA·peptide
interactions. The binding of tRNAfMet to amino-terminal
peptides was also observed using fluorescence-labeled tRNAs and
circular dichroism. These results suggest that a small peptide can bind
to tRNA selectively and that evolution of class II tRNA synthetases may
involve structural changes of amino-terminal extensions for enhanced
selective binding of tRNA.
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INTRODUCTION |
Aminoacyl-tRNA synthetases catalyze the covalent attachments of
amino acids to cognate tRNAs in the first step of protein biosynthesis.
Extensive studies of the structure and function of this family of
enzymes have provided excellent understanding of fundamental principles
of RNA-protein interactions and structures of synthetases. Almost all
synthetases contain a core catalytic domain carrying out adenylation of
an amino acid and an anti-codon binding domain essential for
aminoacylation of cognate tRNA. The core catalytic domains in class I
synthetases resemble dinucleotide binding folds and reside in the amino
termini, whereas a seven-stranded anti-parallel sheet with three
-helices characterizes active sites in class II synthetases and
locates in the carboxyl termini of synthetases.
Beyond the basic amino- and carboxyl-terminal catalytic domains,
eukaryotic and mammalian synthetases (1-3) have evolved idiosyncratic
extensions dispensable for aminoacylation of tRNA. Additionally,
extensive association of synthetases occurs in high eukaryotic
organisms; thus, 9 of the 20 synthetases associate as a multienzyme
complex, ProRS and GluRS form a fused GluProRS (4, 5), and ValRS
associates with the EF1H as a separate complex (6, 7). The
function of the extensions in synthetases has attracted more attention
recently. Controlled proteolysis or hydrophobic interaction
chromatography dissociates several synthetases from the synthetase
complex without significantly affecting enzymatic activities (8, 9);
thus, the extensions in mammalian synthetases likely play pivotal roles
in the structural organization of the synthetase complex. Extensions in
synthetases can be involved in RNA binding (10, 11) or function as
cytokines (12-14). It appears that extensions in synthetases are
multifunctional in at least some cases. Multienzyme complexes of
aminoacyl-tRNA synthetases also provide a model for studying
organization of cellular protein biosynthetic machinery (15, 16). The
primary and quaternary structures of the protein components and the
gross structure of the synthetase complex are well established, but the
structural and functional organization of the synthetase complex is not
well understood.
AspRS1 is a class II
synthetase, its primary structures from more than 50 sources have been
determined, and the three-dimensional structures from bacteria and
yeast have been resolved (17, 18). Mammalian AspRS occurs as the
smallest synthetase in the multisynthetase complex and has been used as
a model for better understanding of the multisynthetase complex and the
extensions in synthetases. Human AspRS (hDRS) has a very short,
32-residue, near neutral extension (19, 20) as opposed to the
71-residue, highly basic extension in the corresponding yeast AspRS
(21). All other mammalian synthetases have longer extensions than AspRS
(1); in particular, IRS has an extension of 180 residues. Deletion of
the amino-terminal extension in mammalian AspRS affects the stability,
the Michaelis-Menten parameters (22), the dimerization (12), and
cellular localization (23, 24), but deletion mutants remain fully
active. Interestingly, the amino-terminal extension reduces the rate of
release of charged tRNA from hDRS (11), and elongation factor 1
with
GTP stimulates the charged tRNA release; thus, the extension in hDRS
directly or indirectly mediates the direct transfer of charged tRNA
from RS to elongation factor 1
(25). The full-length hDRS has
appreciably lower Km to tRNA than that of the
truncated hDRS (22). Altogether, the results of the steady-state and
single-turnover kinetic analyses suggest that the hDRS extension likely
interacts with tRNA or the active site in a manner that may provide
additional understanding of the process of synthetase·RNA
interactions. Furthermore, in contrast to the lysine-rich extensions
described in several recent reports (10, 26, 27), the 32-residue
extension in hDRS is short and is not highly basic. In this paper, we
focus on the tRNA binding by the amino-terminal extension in hDRS using fluorescence spectroscopy and circular dichroism (CD). We report that
the hDRS extension indeed binds to tRNA and magnesium ion plays an
essential role in this case of a neutral nonlysine-rich extension.
Preliminary reports of this study have appeared previously (28).
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EXPERIMENTAL PROCEDURES |
Unfractionated Escherichia coli tRNA, yeast tRNA, and
E. coli tRNAfMet were obtained from Roche
Molecular Biochemicals. Rabbit reticulocytes were purchased from
Green Hectare. The synthetase complex was purified from rabbit
reticulocytes as described previously (29). Ethidium bromide,
trifluoroethanol, octadecaphosphate, soluble calf thymus DNA, and
ribonuclease were obtained from Sigma. Poly(A), poly(U), poly(dA), and
poly(dA-dT) were purchased from Amersham Pharmacia Biotech. The
full-length (hDRS) and the amino-terminal 32-residue-deleted
(hDRS
32) forms of human AspRS were expressed in bacteria as
glutathione S-transferase fusion proteins, subsequently thrombin-digested, and purified as free synthetase as described previously (22). The sequence of the 21-mer synthetic peptide, hDRS
(Thr5-Lys25), was
AcTQRKSQEKPREIMDAAEDYAK-amide, and the sequence of the hexadecamer
peptide, hDRS (Asp12-Arg27), was
AcDPREIMDAAEDYAKER-amide. Both peptides were synthesized and purified
using RPC-C18 high pressure liquid chromatography to be more than 95%
pure by Peptide Technology (Bethesda, MD). The molecular weights of
hDRS (Thr5-Lys25) and hDRS
(Asp12-Arg27) were 2535 (expected 2535) and
1950 (expected 1950), respectively, as determined by mass spectrometry.
Steady-state fluorescence measurements were carried out using a Hitachi
PerkinElmer Life Sciences MPF2A spectrofluorimeter and 0.4- × 0.4-cm2 Hellma quartz microcuvettes at room temperature as
described previously (30). Fluorescence titrations were performed by
adding 1- to 5-µl aliquots to an initial volume of 250 µl in a
standard buffer containing 10 mM Tris-HCl (pH 7.5) and 4 mM MgCl2 unless specified otherwise. Samples
for fluorescence measurements had absorbance < 0.05 absorbance
unit at the excitation wavelength. The extents of decrease of
fluorescence intensity due to inner filter effect were determined by
carrying out parallel titrations with tyrosine or tryptophan at the
same molar concentration as that of the peptide or protein. The net
extents of quenching were used for calculating binding constants.
CD measurements were carried out using a Jasco700 CD spectrophotometer
under nitrogen at 10 p.s.i. as described previously (11). The
wavelengths were calibrated using camphor sulfate. The spectra were
recorded using 1-cm cells and scanned at least three times from 190 to
300 nm. CD titrations were carried out by adding 1-µl aliquots to an
initial volume of 300 µl in a standard buffer of 10 mM
potassium phosphate (pH 7.5) and 4 mM MgCl2
unless specified otherwise. CD spectra were deconvoluted using the
convex constraint analysis (31) to resolve the five fundamental
component CD spectra for various secondary structures of a given sample and 25 proteins with known secondary structures using software supplied
by Jasco.
Binding of tRNA to peptides was analyzed by fluorescence
quenching at varying concentration of peptides (
F) and
saturating concentration of peptides (
Fmax).
Combining these values with the equation of conservation of mass and
the Scatchard equation, Eq. 1 can be derived:
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(Eq. 1)
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where R0, P0,
n, and Kd are the total concentration of
tRNA, the total concentration of peptide, the binding stoichiometry, and the dissociation constant, respectively. Eq. 1 was used instead of
the simple hyperbolic equation to include correction of the total
concentration of peptide to the concentration of unbound peptide.
Nonlinear least square fit was carried out using Sigma Plot. CD
titration was similarly analyzed except the net increases in
ellipticity above the sum of tRNA and peptide ellipticities were used
by subtraction of ellipticities of peptide and tRNA alone from those of
mixtures of peptide and tRNA.
Binding of Mg2+ to tRNA was analyzed by fluorescence
quenching of tRNA-bound ethidium by Mg2+ at varying
concentrations of Mg2+ (
F) and at saturating
concentration of Mg2+ (
Fmax). The
Hill equation (Eq. 2) was used to obtain Hill coefficients:
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(Eq. 2)
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where [Mg2+], h, n, and
Kd are the total concentration of MgCl2,
the Hill coefficient, the stoichiometry, and the dissociation constant, respectively.
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RESULTS |
Binding of tRNAfMet to hDRS and hDRS
32--
The
amino-terminal extension in hDRS dramatically reduces the rate of
release of charged tRNA from hDRS (11), suggesting that tRNA interacts
directly or indirectly with the amino-terminal extension in hDRS.
Noncognate tRNAfMet from E. coli was used to
examine tRNA binding capability of the amino-terminal extension in
hDRS. Noncognate tRNA was used, because the relatively small
amino-terminal extension was not expected to exhibit tRNA identity and
noncognate tRNA should not bind to the cognate tRNA binding site. As
shown in Fig. 1, the full-length hDRS
bound to tRNAfMet as judged by the quenching of the
intrinsic fluorescence of hDRS, and an apparent dissociation constant
of tRNAfMet to hDRS of 0.27 µM was obtained
based on fluorescence titration. No fluorescence quenching of hDRS by
tRNAfMet was observed in the absence of MgCl2
suggesting Mg2+ is essential in the noncognate tRNA
binding. The linear decrease of the fluorescence in hDRS
32 was due
to the UV absorption by tRNA, and the negligible fluorescence quenching
of hDRS
32 by tRNAfMet precluded determination of a
binding constant. The large difference in the tRNAfMet
affinity between hDRS and hDRS
32 suggests that the amino-terminal extension is needed for hDRS to bind to noncognate tRNA.

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Fig. 1.
Fluorescence titration of hDRS and
hDRS 32 with tRNAfMet. The
fluorescence titrations of the intrinsic fluorescence of hDRS ( ) and
hDRS 32 ( ) with E. coli tRNAfMet were
carried in 10 mM Tris-HCl (pH 7.5), 4 mM
MgCl2, and at room temperature by monitoring the
fluorescence intensity at 340 nm ( ex = 275 nm). The
fitted function using Eq. 1 gave a dissociation constant of 0.27 µM for hDRS. The linear decrease of fluorescence
intensity of hDRS 32 resulted from the UV absorption of tRNA.
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Binding of the 21-mer Peptide hDRS
(Thr5-Lys25) to tRNAfMet--
The
binding of tRNAfMet to hDRS could result from interaction
of the amino-terminal extension with the active site or attribute to a
direct binding of tRNA by the extension. The interaction of
tRNAfMet with the amino-terminal extension in hDRS was next
investigated using synthetic peptides. A 21-residue peptide hDRS
(Thr5-Lys25) that contains a putative
amphiphilic helix in the amino-terminal extension in hDRS was
synthesized. The fluorescence intensity of ethidium enhances over
20-fold upon binding to a single strong binding site in tRNA (32, 33),
and the fluorescence of tRNA-bound ethidium was monitored to assess
binding of tRNA to amino-terminal peptides. As shown in Fig.
2, the fluorescence of tRNA-bound
ethidium was quenched 25% by hDRS (Thr5-Lys25)
at 4 mM MgCl2. In the absence of
MgCl2, on the contrary, the fluorescence of tRNA-bound
ethidium was not at all affected by the peptide. Free ethidium
fluorescence in the absence of tRNA was not affected by the peptide
under the same conditions either. Fluorescence titration of
tRNAfMet and hDRS (Thr5-Lys25) gave
an apparent dissociation constant of 1.7 µM. The number of peptide binding sites in tRNA was determined by Job analysis (34,
35), in which the fluorescence intensities were determined at varying
molar ratios of the peptide to tRNA without changing their total molar
concentration. As shown in Fig. 2, the maximal change was obtained when
tRNA and the peptide were present at a 1:1 molar ratio; thus, there is
one binding site per tRNA molecule.

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Fig. 2.
Fluorescence titration of
tRNAfMet-bound ethidium with hDRS
(Thr5-Lys25). Fluorescence titrations of
tRNAfMet were carried out in 10 mM Tris-HCl, pH
7.5, in the absence ( ) and the presence ( ) of 4 mM
MgCl2 by monitoring the fluorescence intensity of
tRNAfMet-bound ethidium at 610 nm ( ex = 470 nm). The fitted function using Eq. 1 gave a dissociation constant
Kd of 1.7 µM. Inset, Job
(34) analysis of the stoichiometry of peptide and tRNA based on
the fluorescence changes of the tRNA-bound ethidium at varying molar
ratios of tRNA to the peptide at the same total molar concentration of
tRNA and peptide.
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When the Tyr fluorescence of the peptide was monitored, a moderate
quenching by tRNAfMet, 11%, was observed after correction
for the inner filter effect. In the absence of MgCl2, the
Tyr fluorescence of the peptide was not quenched by
tRNAfMet.
When the CD spectrum of the peptide was compared with that of the
peptide in the presence of tRNA after subtraction of CD of tRNA alone,
significant changes of the peptide CD between 200 nm and 250 nm were
observed (Fig. 3). Comparison of the CD
spectra of tRNA in the absence and in the presence of the peptide
(after subtraction of CD of peptide) indicated that no detectable
changes in the spectrum between 250 nm and 300 nm where tRNA absorbs. Again, no CD changes of the peptide or tRNA were found in the absence
of MgCl2. An apparent dissociation constant of 0.9 µM was obtained by monitoring ellipticity at 208 nm in
the binding of tRNAfMet to the peptide.

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Fig. 3.
CD spectra of free and tRNA-bound hDRS
(Thr5-Lys25) peptide. The CD spectrum of
the peptide was obtained in 10 mM potassium phosphate, pH
7.5, and 4 mM MgCl2. The CD spectrum of the
free peptide ( ) is superimposed on the CD spectrum of the peptide in
complex with tRNA ( ) that was obtained by subtracting the CD
spectrum of tRNAfMet from the CD spectrum of
tRNAfMet in the presence of a stoichiometric amount of the
peptide.
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The binding of the peptide to tRNAfMet appears to be
selective to tRNA among polyanions and polynucleotides. As shown in
Table I, the fluorescence of
poly(dA-dT)-bound ethidium was not affected by the peptide hDRS
(Thr5-Lys25). The Tyr fluorescence of hDRS
(Thr5-Lys25) was also monitored for binding to
nucleic acids other than tRNA. As shown in Table I, tyrosine
fluorescence quenching was small for poly(A), poly(U), poly(dA-dT), or
an oligodeoxynucleotide. The tyrosine fluorescence of the hDRS
(Thr5-Lys25) was not at all affected by
polyphosphate (Table I). Finally, the ellipticity of the peptide was
monitored to detect the binding of the peptide to polyphosphate and
oligodeoxynucleotides. No change in the CD spectrum of the peptide was
observed upon addition of polyphosphate. When the CD spectra of the
peptide were compared in the absence and presence of the
oligodeoxynucleotide after correction of the oligodeoxynucleotide CD,
no changes in the CD spectrum of the peptide were found.
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Table I
Binding of hDRS(T5-K25) to tRNAfMet and polyanions
All fluorescence measurements were carried out using 0.2 mg/ml peptide
hDRS (Thr5-Lys25) in 10 mM Tris-HCl (pH
7.5) and 4 mM MgCl2. The intrinsic fluorescence of
the peptide was characteristic of that of Tyr. The Tyr fluorescence
changes were corrected for the inner filter effect of tRNA. The
concentrations of all polynucleotides were adjusted to 0.1 absorbance
unit at 260 nm for comparison. The ethidium concentration was 1.4 µM and the ethidium fluorescence was monitored at 610 nm
and excited at 470 nm. In the absence of MgCl2, no changes in
either ethidium or Tyr fluorescence were observed with either
tRNAfMet or a polyanion.
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Binding of the Peptide to Other Fluorescence-labeled
tRNAs--
Modified tRNAfMet labeled with single
fluorescence probe at the 3'-end, S8-C13, and D-20 with fluorescein,
photo-cross-link, and ethidium, respectively (30), were examined for
binding to hDRS (Thr5-Lys25). The fluorescence
in tRNAfMet (3'-fluorescein), tRNAfMet (8-13),
and tRNAfMet (D-ethidium) was monitored separately
upon addition of the peptide hDRS (Thr5-Lys25).
As shown in Fig. 4, the fluorescence in
tRNAfMet (8-13) was quenched by 20%, whereas few changes
in the fluorescence of tRNAfMet (3'-fluorescein) or
tRNAfMet (D-ethidium) were observed. In the absence of
MgCl2, the peptide had no effect on the fluorescence of
tRNAfMet (8-13).

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Fig. 4.
Fluorescence titration of modified
tRNAfMet with the peptide. The reduced,
photo-cross-linked tRNAfMet (8-13) ( ) was titrated with
the hDRS (Thr5-Lys25) peptide in 10 mM Tris-HCl (pH 7.5), 4 mM MgCl2.
The fluorescence intensity at 436 nm ( ex = 388 nm) was
monitored. The fitted line was obtained using Eq. 1 and a
dissociation constant Kd of 3.1 µM.
Titration of fluorescent-labeled tRNAfMet (D-ethidium)
( ) and tRNAfMet (3'-fluorescein) ( ) with the peptide
was carried out under the same conditions.
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The binding of hDRS (Thr5-Lys25) to
tRNAfMet was analyzed at varying concentrations of ethidium
to determine whether hDRS (Thr5-Lys25) and
ethidium bind competitively to tRNAfMet. The binding of the
hDRS (Thr5-Lys25) to tRNAfMet was
determined at varying concentrations of ethidium in 10 mM Tris-HCl (pH 7.5) and 4 mM MgCl2, and the
fitted titration curves using Eq. 1 gave apparent dissociation
constants, 1.7, 1.86, 3.5, and 23.4 µM, at 1.4, 2.8, 5.6 and 11.2 µM ethidium, respectively. The amino-terminal
peptide evidently showed weaker binding to tRNA at higher
concentrations of ethidium; however, the effect of ethidium on the
peptide binding to tRNA could not be modeled by simple competitive binding.
Roles of MgCl2 on tRNA Binding--
Mg2+
was needed for the binding of the amino-terminal peptide to
tRNAfMet as demonstrated by fluorescence quenching and CD
changes. Divalent cations such as Ca2+ and Zn2+
or the monovalent cation Na+ could not substitute for
Mg2+ in the binding of the peptide to tRNAfMet
as judged by the extent of quenching of the fluorescence of tRNA-bound ethidium by the peptide. The roles of Mg2+ ion in the
interaction of tRNA and the peptide could be maintaining the proper
conformation of tRNA or mediating the formation of the complex.
To assess the role of Mg2+ in the binding of the peptide to
tRNA, fluorescence titration of the tRNA-bound ethidium with the peptide was carried out at varying concentrations of MgCl2.
The effect of Mg2+ may be represented by the mass action of
the process:
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(Eq. 3)
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When Ka is defined in terms of the equilibrium
concentrations of tRNA, peptide and the complex, then it is readily shown that
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(Eq. 4)
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where
n is the difference of the average numbers of
Mg2+ bound in free tRNA and peptide-bound tRNA. The
apparent binding constants of the peptide to tRNA,
Ka, increased with increasing concentrations of
MgCl2. The linear relationship of the plot of log
Ka versus log [Mg2+] gave a
slope of 1.33 (Fig. 5). A slope of
greater than one suggests at least one additional Mg2+ ion
bound in the tRNA·peptide complex upon the formation of the complex
as compared with free tRNA.

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Fig. 5.
Dependence of the apparent association
constant for the peptide binding to tRNA at varying concentrations of
magnesium ion. Fluorescence titration of the tRNA-bound ethidium
with the hDRS (Thr5-Lys25) peptide at 0.5, 2, 4, and 10 mM MgCl2 was carried as described in
Fig. 2. The apparent association constants of peptide to tRNA (in
µM) were determined, and the logs of apparent association
constants were plotted against the logs of the concentration of
MgCl2. The fitted line represents the linear
least-square fit to the data.
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The association of Mg2+ with tRNA was next analyzed by
monitoring the fluorescence changes of tRNAfMet-bound
ethidium by Mg2+. As shown in Fig.
6, the binding of Mg2+ is
highly cooperative and the binding isotherm gave a Hill coefficient of
3.4. This is in good agreement with earlier studies (36). When the
association of Mg2+ with tRNAfMet was similarly
analyzed in the presence of a saturating amount of the 21-mer peptide,
a Hill coefficient of 4.5 was obtained.

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Fig. 6.
Association of Mg2+ with tRNA in
the presence of the peptide. The fluorescence of tRNA-bound
ethidium was monitored upon addition of MgCl2 in the
absence (inset) and the presence of the 21-mer peptide. The
fitted lines were obtained by nonlinear least-square fit to
Hill equation. The fitted function gave Hill coefficients of 3.4 and
4.5 in the absence and the presence of the hDRS
(Thr5-Lys25) peptide, respectively.
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When the effect of NaCl on the binding of the peptide to tRNA in the
presence of MgCl2 was analyzed, in contrast to the
stimulatory effect of Mg2+, the extent of fluorescence
quenching decreased at increasing concentrations of NaCl. The apparent
binding constants of the peptides to tRNAfMet also
decreased at increasing concentrations of NaCl and gave a linear
relationship of log Ka versus
log[NaCl] with a slope of
0.2.
Binding of the 16-mer Peptide hDRS
(Asp12-Arg27) to tRNAfMet--
A
shorter 16-mer peptide in the amino-terminal extension of hDRS was also
examined. The peptide hDRS (Asp12-Arg27), which
has an additional acetylated Asp as a helix cap and an additional basic
Arg residue at the carboxyl terminus, has significantly higher
propensity of forming helical secondary structure than hDRS (T5-25)
(11). The binding of tRNAfMet by the peptide hDRS
(Asp12-Arg27) was similarly analyzed. As shown
in Fig. 7, the fluorescence of ethidium
noncovalently bound to tRNAfMet was similarly quenched by
hDRS (Asp12-Arg27), showing an apparent
dissociation constant of 8.9 µM. Again, in the absence of
Mg2+, no change of the ethidium fluorescence was
observed.

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Fig. 7.
Binding of the 16-mer peptide to
tRNAfMet. The fluorescence intensity of
tRNAfMet-bound ethidium was monitored at 610 nm
( ex = 470 nm). Fluorescence measurements were
carried out in 10 mM Tris-HCl (pH 7.5) in the absence ( )
and the presence ( ) of 4 mM MgCl2. The
fitted function using Eq. 1 gave a dissociation constant
Kd of 8.9 µM.
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Molecular Model of the Peptide·tRNA Complex--
Although the
three-dimensional structures of yeast and bacteria AspRS have been
determined, the amino-terminal extension was mobile and its structure
so far remains unresolved. The conformation of the peptide was first
modeled based on its 48% sequence identity to Salmonella
typhimurium glutamine synthetase () in the Rutgers Protein
Data Base. Coordinates of the peptide backbone were assigned to the
peptide according to the coordinates of the reference glutamine synthetase (PDB 2LGS). The energy of the structure was minimized using
the DISCOVER Homology module from Biosym by running 1000 dynamics
steps followed by 100 equilibration steps at 300 °K and 1-ps
time step. The dynamics process was followed by 3000 energy minimization steps. The lowest energy was
118 kcal/mol, and the predicted secondary structure had 11 hydrogen bonds. The peptide was
then docked, using the DISCOVER Affinity module, onto
tRNAPhe in the Rutgers Protein Data Base with all possible
orientations and 100 iterations while the intermolecular energy between
the peptide and tRNA was monitored. The lowest energy among docked structures was
99 kcal/mol. Binding of the peptide to tRNA involved conformational changes in the peptide and resulted in a slightly higher
disordered secondary structure than that in the free peptide. A stereo
view of the backbone model of the complex of hDRS
(Thr5-Lys25) with tRNAPhe is shown
in Fig. 8.

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Fig. 8.
Molecular model of the tRNA·hDRS
(Thr5-Lys25) complex. The backbone form of
tRNA is shown in complex with hDRS (Thr5-Lys25)
with amino and carboxyl termini in proximity of the acceptor and
anti-codon stems, respectively.
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DISCUSSION |
Nonspecific tRNA Binding by the Amino-terminal Extension of a Class
II Synthetase--
The present study shows that the amino-terminal
peptide in hDRS binds to tRNA based on fluorescence and CD changes in
tRNAfMet or the peptide. Ethidium was used as an extrinsic
fluorescence probe in analyzing the binding of tRNA by the peptide, and
increasing the concentration of ethidium was found to reduce the
affinity of tRNA to the peptide. A tighter binding to tRNA by the
peptide can be expected in the absence of ethidium under the same
conditions. Indeed, a slightly higher apparent binding constant based
on the ellipticity at 208 nm was observed as compared with those
obtained using fluorescence-labeled tRNA or ethidium-bound tRNA. Both
fluorescence and CD results demonstrated the binding of tRNA by the
peptide. The binding appears to be selective to tRNA, because other
polynucleotides and polyphosphate did not bind to the synthetic
peptide. These results are in notable contrast to the amino-terminal
peptide in yeast DRS. Polyphosphate and DNA bind indiscriminately to
the highly basic extension in yeast DRS (37). It appears that the amino-terminal extension in DRS evolved from a highly basic extension in yeast to a nearly neutral extension in mammals and resulted in its
selective binding to tRNA. To our knowledge, this is the first time an
amino-terminal peptide was found to bind tRNA selectively. Several
nonspecific tRNA binding domains have been found recently in GlnRS
(26), AspRS (10), and AlaRS (27) and are characterized by clusters of
basic residues in the tRNA binding domains. The nonspecific RNA binding
domains evidently facilitate the formation of a specific and essential
tRNA-protein complex and optimize recognition of tRNA. In addition, a
zinc binding domain distal to the tRNA-binding site in IRS is essential
in tRNA binding (38). The mechanisms of tRNA recognition through these
RNA binding domains in synthetases are yet to be elucidated.
Roles of Magnesium Ion and Electrostatic
Interaction--
Mg2+ mediates and enhances the binding of
tRNA by the amino-terminal peptides in hDRS. On the basis of the slope
of the log Ka versus log
[MgCl2] plot, there is clearly at least one additional
Mg2+ in the tRNA·peptide complex than that in free tRNA.
Mg2+ binds cooperatively to tRNA and stabilizes the
conformation of tRNA. The observation that the Hill coefficient
increased to 4.5 from 3.4 in the presence of the peptide is consistent
with the increase of Mg2+ sites, although it should be
cautioned that the Hill coefficient could have changed without changing
the number of binding sites. A cluster of four acidic amino acids
(Glu15, Asp18, Glu21,
Asp22) in hDRS (Thr5-Lys25) could
be involved in the binding of Mg2+ and mediate the binding
of the phosphate-ribose backbone in RNA. No Mg2+
requirement or Mg2+-mediated binding of tRNA by the highly
basic or lysine-rich peptides has been reported, suggesting a different
mode of RNA binding is utilized by neutral or nonlysine-rich
peptides. Whether such mode of interaction is general for other neutral
extensions or RNA binding domains is yet to be explored.
Na+ cannot replace Mg2+ in facilitating the
binding of the peptide to tRNA, and Na+ actually reduces
tRNA affinity to the peptide. The slope of log Ka
versus log [Na+] was small and negative in
comparison to those such as drug·DNA interactions dominated by
electrostatic interaction. The apparent dissociation constants of hDRS
(Thr5-Lys25) and the 16-mer to
tRNAfMet were found to be 1.7 and 8.9 µM,
respectively. Three basic residues in hDRS
(Thr5-Lys25) were absent in the 16-mer; thus,
additional basic residues in hDRS (Thr5-Lys25)
moderately enhanced affinity to tRNA. It appears that electrostatic interactions between tRNA and the basic residues in the extension may
complement the roles of Mg2+ in tRNA binding.
Conformation of the Peptide·tRNA Complex--
The
molecular model of the peptide, which showed a disordered amino half of
the peptide before Pro and a partial helical carboxyl half after Pro,
is in general consistent with previously reported CD analyses. CD of a
22-mer, hDRS (T5-E26), and a 16-mer, hDRS (Asp12-Arg27), under various conditions
indicated hDRS (Asp12-Arg27) has a high
propensity of forming helical secondary structure and hDRS (T5-E26) has
a low propensity (11). In the presence of trifluoroethanol, hDRS
(Asp12-Arg27) exhibited up to 61% helical
content. However, the model must be considered tentative. Molecular
modeling of the tRNA·peptide complex suggests that the peptide is
preferentially located at the inside surface of the L-shaped tRNA with
proximity to the acceptor and anti-codon stems. Such a model is
consistent with the experimental data but must be considered tentative.
It is intriguing that the binding of the peptide to tRNA is inhibited by ethidium, considering that the ethidium binding site is near the
base of the acceptor stem as localized by singlet-singlet energy
transfer experiments (39). Because binding of ethidium to tRNA could be
nonintercalative and nonfluorogenic, the nature of the
competition of the peptide with ethidium remains to be clarified;
nonetheless, the quenching of fluorescence of ethidium in
tRNAfMet is consistent with the suggestion that the peptide
binds near the base of the acceptor stem. The quenching of the
fluorescence of tRNA (8-13) and unchanged fluorescence in tRNA labeled
at the 3'-end and D20 by hDRS (Thr5-Lys25) are
also consistent with the model of the peptide and tRNA.
The Multisynthetase Complex and the Synthetase Extensions--
The
present results support the likelihood that the evolution of extensions
in mammalian synthetases may facilitate transferring tRNA between the
periphery of the enzyme and the active site of the enzyme. The ability
of tRNA binding by the amino-terminal extension in hDRS is in accord
with its control of the release of charged tRNA (25). The
amino-terminal extension in hDRS also reduces the tRNA Michaelis-Menten
constant as compared with hDRS
32 (22). Similarly, the nonspecific
tRNA binding extension in yeast AspRS decreases the tRNA
Michaelis-Menten constant (10). Clearly, fusion of a nonspecific tRNA
binding domain to either class I or class II synthetase significantly
enhances synthetase·tRNA interactions in terms of affinity and
specificity (10, 26, 27).
The RNA binding capability of extensions in synthetases can conceivably
facilitate not only the intramolecular transfer of RNA but also
intermolecular transfers of RNA from one protein to another. The
amino-terminal extension in hDRS mediates the direct transfer of
charged tRNA from hDRS to the elongation factor 1
(25). Such
intermolecular transfer is likely to play roles in the observed effects
of yeast Arc1p on MetRS (40, 41) or a human homologue, p43, on ArgRS
(42). The detailed mechanisms of action and the tRNA binding motifs in
these cases are yet to be determined.
Highly charged and potentially amphiphilic helices are prevalent in
mammalian synthetases but were absent in bacterial synthetases (2). The
highly basic extensions in yeast synthetases endowed the yeast
synthetases with the ability to bind polyanions (37). Some mammalian
synthetases such as Val- and LysRSs also contain such highly basic
extensions (43, 44). The present results open the possibility that the
potentially amphiphilic helices in other mammalian synthetases may be
involved in the RNA·synthetase interactions as well.
Better understanding of the RNA·peptide interactions provides models
of RNA·protein interactions and should be useful in the elucidation
of the mechanisms in the transport of RNA from one protein to another
and in the intracellular transport of RNA (45) as well as in protein
biosynthesis (46).
 |
ACKNOWLEDGEMENT |
We thank Dr. P. Boon Chock (National
Institutes of Health) for many helpful discussions.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Tel.: 202-687-6090;
Fax: 202-687-6209; E-mail: yangdc@georgetown.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007570200
 |
ABBREVIATIONS |
The abbreviations used are:
AspRS, aspartyl-tRNA
synthetase;
DRS, AspRS;
hDRS, human AspRS;
RS, tRNA synthetase;
CD, circular dichroism.
 |
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