Magnesium Ion-mediated Binding to tRNA by an Amino-terminal Peptide of a Class II tRNA Synthetase*

Rasha Hammamieh and David C. H. YangDagger

From the Department of Chemistry, Georgetown University, Washington, DC 20057

Received for publication, August 18, 2000, and in revised form, September 7, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 1alpha ; 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 1alpha 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 1alpha (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).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (hDRSDelta 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 (Delta F) and saturating concentration of peptides (Delta Fmax). Combining these values with the equation of conservation of mass and the Scatchard equation, Eq. 1 can be derived:
&Dgr;F=&Dgr;F<SUB>max</SUB><FR><NU>(nR<SUB>0</SUB>+P<SUB>0</SUB>+K<SUB>d</SUB>)−[(nR<SUB>0</SUB>+P<SUB>0</SUB>+K<SUB>d</SUB>)<SUP>2</SUP>−4nR<SUB>0</SUB>P<SUB>0</SUB>]<SUP>1/2</SUP></NU><DE>2R<SUB>0</SUB></DE></FR>, (Eq. 1)
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+ (Delta F) and at saturating concentration of Mg2+ (Delta Fmax). The Hill equation (Eq. 2) was used to obtain Hill coefficients:
&Dgr;F=&Dgr;F<SUB>max</SUB><FR><NU>n[<UP>Mg<SUP>2+</SUP></UP>]<SUP>h</SUP></NU><DE>K<SUB>d</SUB>+[<UP>Mg<SUP>2+</SUP></UP>]<SUP>h</SUP></DE></FR>, (Eq. 2)
where [Mg2+], h, n, and Kd are the total concentration of MgCl2, the Hill coefficient, the stoichiometry, and the dissociation constant, respectively.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of tRNAfMet to hDRS and hDRSDelta 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 hDRSDelta 32 was due to the UV absorption by tRNA, and the negligible fluorescence quenching of hDRSDelta 32 by tRNAfMet precluded determination of a binding constant. The large difference in the tRNAfMet affinity between hDRS and hDRSDelta 32 suggests that the amino-terminal extension is needed for hDRS to bind to noncognate tRNA.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Fluorescence titration of hDRS and hDRSDelta 32 with tRNAfMet. The fluorescence titrations of the intrinsic fluorescence of hDRS (open circle ) and hDRSDelta 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 (lambda 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 hDRSDelta 32 resulted from the UV absorption of tRNA.

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.



View larger version (23K):
[in this window]
[in a new window]
 
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 (open circle ) and the presence () of 4 mM MgCl2 by monitoring the fluorescence intensity of tRNAfMet-bound ethidium at 610 nm (lambda 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.

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.



View larger version (20K):
[in this window]
[in a new window]
 
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 (triangle ) 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.

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.


                              
View this table:
[in this window]
[in a new window]
 
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.

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).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Fluorescence titration of modified tRNAfMet with the peptide. The reduced, photo-cross-linked tRNAfMet (8-13) (black-square) 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 (lambda 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) (open circle ) and tRNAfMet (3'-fluorescein) () with the peptide was carried out under the same conditions.

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:
<UP>tRNA + peptide + &Dgr;</UP>n<UP>Mg<SUP>2+</SUP></UP> ⇄ [<UP>tRNA · Mg · peptide</UP>] (Eq. 3)
When Ka is defined in terms of the equilibrium concentrations of tRNA, peptide and the complex, then it is readily shown that
<FENCE><FR><NU>&dgr; <UP>log</UP>K<SUB><UP>a</UP></SUB></NU><DE><UP>&dgr; log</UP>[<UP>Mg<SUP>2+</SUP></UP>]</DE></FR></FENCE><SUB>T,P</SUB>=&Dgr;n (Eq. 4)
where Delta 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.



View larger version (15K):
[in this window]
[in a new window]
 
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.

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.



View larger version (18K):
[in this window]
[in a new window]
 
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.

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.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Binding of the 16-mer peptide to tRNAfMet. The fluorescence intensity of tRNAfMet-bound ethidium was monitored at 610 nm (lambda ex = 470 nm). Fluorescence measurements were carried out in 10 mM Tris-HCl (pH 7.5) in the absence (open circle ) and the presence () of 4 mM MgCl2. The fitted function using Eq. 1 gave a dissociation constant Kd of 8.9 µM.

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.



View larger version (20K):
[in this window]
[in a new window]
 
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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 hDRSDelta 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 1alpha (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.

Dagger 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Mirande, M. (1991) Prog. Nucleic Acid Res. Mol. Biol. 40, 95-142[Medline] [Order article via Infotrieve]
2. Yang, D. C. H. (1996) Curr. Topics Cell. Regul. 34, 101-136[Medline] [Order article via Infotrieve]
3. Kisselev, L. L., and Wolfson, A. D. (1994) Prog. Nucleic Acid Res. Mol. Biol. 48, 83-142[Medline] [Order article via Infotrieve]
4. Fett, R., and Knippers, R. (1991) J. Biol. Chem. 266, 1448-1455[Abstract/Free Full Text]
5. Cerini, C., Kerjan, P., Astier, M., Gratecos, D., Mirande, M., and Semeriva, M. (1991) EMBO J. 10, 4267-4277[Abstract]
6. Motorin, Y. A., Wolfson, A. D., Orlovsky, A. F., and Gladilin, K. L. (1988) FEBS Lett. 238, 262-264[CrossRef][Medline] [Order article via Infotrieve]
7. Bec, G., and Waller, J. P. (1989) J. Biol. Chem. 264, 21138-21145[Abstract/Free Full Text]
8. Cirakoglu, B., and Waller, J. P. (1985) Eur. J. Biochem. 151, 101-110[Abstract]
9. Johnson, D. L., Dang, C. V., and Yang, D. C. H. (1980) J. Biol. Chem. 255, 4362-4366[Abstract/Free Full Text]
10. Frugier, M., Moulinier, L., and Giege, R. (2000) EMBO J. 19, 2371-2380[Abstract/Free Full Text]
11. Reed, V. S., and Yang, D. C. H. (1994) J. Biol. Chem. 269, 32937-32941[Abstract/Free Full Text]
12. Agou, F., Waller, J.-P., and Mirande, M. (1996) J. Biol. Chem. 271, 29295-29303[Abstract/Free Full Text]
13. Wakasugi, K., and Schimmel, P. (1999) J. Biol. Chem. 274, 23155-23159[Abstract/Free Full Text]
14. Kleeman, T. A., Wei, D., Simpson, K. L., and First, E. A. (1997) J. Biol. Chem. 272, 14420-14425[Abstract/Free Full Text]
15. Rho, S. B., Kim, M. J., Lee, J. S., Seol, W., Motegi, H., Kim, S., and Shiba, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4488-4493[Abstract/Free Full Text]
16. Quevillon, S., Robinson, J. C., Berthonneau, E., Siatecka, M., and Mirande, M. (1999) J. Mol. Biol. 285, 183-195[CrossRef][Medline] [Order article via Infotrieve]
17. Sauter, C., Lorber, B., Cavarelli, J., Moras, D., and Giege, R. (2000) J. Mol. Biol. 299, 1313-1324[Medline] [Order article via Infotrieve]
18. Ruff, M., Krishnaswamy, S., Boeglin, M., Poterszman, A., Mitschler, A., Podjarny, A., Rees, B., Thierry, J. C., and Moras, D. (1991) Science 252, 1682-1689[Medline] [Order article via Infotrieve]
19. Jacobo-Molina, A., Peterson, R., and Yang, D. C. H. (1989) J. Biol. Chem. 264, 16608-16612[Abstract/Free Full Text]
20. Mirande, M., and Waller, J. P. (1989) J. Biol. Chem. 264, 842-847[Abstract/Free Full Text]
21. Sellami, M., Prevost, G., Bonnet, J., Dirheimer, G., and Gangloff, J. (1986) Gene (Amst.) 40, 349-352
22. Escalante, C., and Yang, D. C. H. (1993) J. Biol. Chem. 268, 6014-6023[Abstract/Free Full Text]
23. Mirande, M., Lazard, M., Martinez, R., and Latreille, M. T. (1992) Eur. J. Biochem. 203, 459-466[Abstract]
24. Escalante, C., Qasba, P. K., and Yang, D. C. H. (1994) Mol. Cell. Biochem. 140, 55-63[Medline] [Order article via Infotrieve]
25. Reed, V. S., Wastney, M., and Yang, D. C. H. (1994) J. Biol. Chem. 269, 32932-32936[Abstract/Free Full Text]
26. Wang, C. C., Morales, A. J., and Schimmel, P. (2000) J. Biol. Chem. 275, 17180-17186[Abstract/Free Full Text]
27. Ribas de Pouplana, L., Buechter, D., Sardesai, N. Y., and Schimmel, P. (1998) EMBO J. 17, 5449-5457[Abstract/Free Full Text]
28. Hammamieh, R., and Yang, D. C. H. (1997) FASEB J. 11, A1024
29. Godar, D. E., Garcia, V., Jacobo, A., Aebi, U., and Yang, D. C. H. (1988) Biochemistry 27, 6921-6928[Medline] [Order article via Infotrieve]
30. Ferguson, B. Q., and Yang, D. C. H. (1986) Biochemistry 25, 2743-2748[Medline] [Order article via Infotrieve]
31. Perczel, A., Park, K., and Fasman, G. D. (1992) Anal. Biochem. 203, 83-93[Medline] [Order article via Infotrieve]
32. Bittman, R. (1969) J. Mol. Biol. 46, 251-268[Medline] [Order article via Infotrieve]
33. Tao, T., Nelson, J. H., and Cantor, C. R. (1970) Biochemistry 9, 3514-3524[Medline] [Order article via Infotrieve]
34. Job, P. (1928) Ann. Chim. (Paris) 9, 113-203
35. Huang, C. Y. (1982) Methods Enzymol. 87, 509-525[Medline] [Order article via Infotrieve]
36. Stein, A., and Crothers, D. M. (1976) Biochemistry 15, 157-160[Medline] [Order article via Infotrieve]
37. Agou, F., Yang, Y., Gesquiere, J.-C., Waller, J.-P., and Guittet, E. (1995) Biochemistry 34, 569-576[Medline] [Order article via Infotrieve]
38. Glasfeld, E., and Schimmel, P. (1997) Biochemistry 36, 6739-6744[CrossRef][Medline] [Order article via Infotrieve]
39. Ferguson, B. Q., and Yang, D. C. H. (1986) Biochemistry 25, 5298-5304[Medline] [Order article via Infotrieve]
40. Simos, G., Segref, A., Fasiolo, F., Hellmuth, K., Shevchenko, A., Mann, M., and Hurt, E. C. (1996) EMBO J. 15, 5437-5448[Abstract]
41. Simos, G., Sauer, A., Fasiolo, F., and Hurt, E. C. (1998) Mol Cell 1, 235-242[Medline] [Order article via Infotrieve]
42. Park, S. G., Jung, K. H., Lee, J. S., Jo, Y. J., Motegi, H., Kim, S., and Shiba, K. (1999) J. Biol. Chem. 274, 16673-16676[Abstract/Free Full Text]
43. Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816[Medline] [Order article via Infotrieve]
44. Shiba, K., Stello, T., Motegi, H., Noda, T., Musier-Forsyth, K., and Schimmel, P. (1997) J. Biol. Chem. 272, 22809-22816[Abstract/Free Full Text]
45. Grosshans, H., Simos, G., and Hurt, E. (2000) J. Struct. Biol. 129, 288-294[CrossRef][Medline] [Order article via Infotrieve]
46. Negrutskii, B. S., Stapulionis, R., and Deutscher, M. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 964-968[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.