Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation
2002; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.1093/emboj/21.4.760
ISSN1460-2075
Autores Tópico(s)Plant Disease Resistance and Genetics
ResumoArticle15 February 2002free access Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation Frank Walter Frank Walter UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Joern Pütz Joern Pütz UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Richard Giegé Richard Giegé UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Eric Westhof Corresponding Author Eric Westhof UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Frank Walter Frank Walter UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Joern Pütz Joern Pütz UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Richard Giegé Richard Giegé UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Eric Westhof Corresponding Author Eric Westhof UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France Search for more papers by this author Author Information Frank Walter1, Joern Pütz1, Richard Giegé1 and Eric Westhof 1 1UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg, Cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:760-768https://doi.org/10.1093/emboj/21.4.760 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Aminoglycosides inhibit translation in bacteria by binding to the A site in the ribosome. Here, it is shown that, in yeast, aminoglycosides can also interfere with other processes of translation in vitro. Steady-state aminoacylation kinetics of unmodified yeast tRNAAsp transcript indicate that the complex between tRNAAsp and tobramycin is a competitive inhibitor of the aspartylation reaction with an inhibition constant (KI) of 36 nM. Addition of an excess of heterologous tRNAs did not reverse the charging of tRNAAsp, indicating a specific inhibition of the aspartylation reaction. Although magnesium ions compete with the inhibitory effect, the formation of the aspartate adenylate in the ATP–PPi exchange reaction by aspartyl-tRNA synthetase in the absence of the tRNA is not inhibited. Ultraviolet absorbance melting experiments indicate that tobramycin interacts with and destabilizes the native L-shaped tertiary structure of tRNAAsp. Fluorescence anisotropy using fluorescein-labelled tobramycin reveals a stoichiometry of one molecule bound to tRNAAsp with a KD of 267 nM. The results indicate that aminoglycosides are biologically effective when their binding induces a shift in a conformational equilibrium of the RNA. Introduction Aminoacylation of transfer RNA (tRNA) is essential for cell replication and growth. It is therefore an attractive target for therapeutic intervention. The following strategies have been explored so far: (i) amino acid analogues (Loftfield, 1973; Vasquez, 1974), (ii) oligonucleotides mimicking tRNA features (Loftfield, 1973), (iii) aminoacyl-adenylate analogues (Sassanfar et al., 1996) and (iv) blocking the formation of initiator tRNAfMet (Loftfield, 1973). Up to now, most of the known natural products targeted against specific aminoacyl-tRNA synthetases (aaRSs), or apparent amino acid specificities of the aaRSs could not be developed into an antibiotic due mainly to their lack of systemic bioavailability (Schimmel et al., 1998). The correct aminoacylation of tRNAs by their cognate synthetase is crucial for the accurate transmission of genetic information. It is determined by specific structural features of the tRNAs (Giegé et al., 1998). Although the global architecture of tRNAs is highly conserved, subtle protein–tRNA interfacial contacts guarantee specific aminoacylation of each tRNA by a specific synthetase. The aminoacylation reaction of yeast tRNAAsp (Figure 1A) by its cognate aspartyl-tRNA synthetase (AspRS) is biochemically (Romby et al., 1985; Pütz et al., 1991; Frugier et al., 1994) and structurally well described (Westhof et al., 1985; Cavarelli et al., 1993). Figure 1.Yeast tRNAAsp and the aminoglycoside tobramycin. (A) Sequence of yeast tRNAAsp transcript (Gangloff et al., 1971) showing the change of the first base pair (U1–A72→G1–C72); nucleotides are numbered according to Sprinzl et al. (1998). Identity nucleotides of the aspartylation reaction are shadowed (Pütz et al., 1991; Frugier et al., 1994). The G1–C72 wild-type transcript shows equivalent aspartylation parameters to those of fully modified tRNAAsp and U1–A72 transcripts (Pütz et al., 1991). (B) Structure of the aminoglycoside antibiotic tobramycin, a member of the 2′deoxystreptamine group. The antibiotics of the aminoglycoside family result from modifications of neamine, a two-ring system made of 2-deoxystreptamine (called ring B or II) glycosylated at the 4-position by a 6-membered amino-sugar (called ring A or I) of the glycopyranoside series. Further modifications with various amino-sugars at the 6-position lead to the kanamycin family. Download figure Download PowerPoint Aminoglycosides are known to interact with ribosomal RNAs inhibiting translation at the ribosome level (Blanchard et al., 1998). They further interfere with translational control (Tok et al., 1999), viral transcription of HIV (Zapp et al., 1993; Mei et al., 1995) and ribozyme activity (reviewed in Schroeder et al., 2000). Here, the interaction of tobramycin, a common aminoglycoside of the 2′deoxystreptamine group (Figure 1B), with the yeast tRNAAsp–AspRS complex was investigated. It is demonstrated that the aminoacylation of tRNAAsp, a primary step in protein synthesis, can be specifically inhibited by tobramycin with binding affinities in the nanomolar range. Tobramycin is not the first compound shown to inhibit aminoacylation. Purpuromycin had already been shown to inhibit the aminoacylation reaction at the level of tRNA charging, but with several molecules of purpuromycin binding with micromolar affinity to all tRNAs (Kirillov et al., 1997). Similarly, tobramycin is not the first compound shown to interfere with an RNA–protein enzymic system. Recently, it has been shown that synthetic benzimidazole derivatives inhibit the processing of the precursor-tRNAs to mature tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA), but with I50 values between 5 and 20 μM (Hori et al., 2001). Results Aspartylation activity of tRNAAsp in the presence of tobramycin Aminoacylation experiments with yeast AspRS (Figure 1) reported here were carried out on molecules obtained in vitro by transcription with T7 RNA polymerase. Figure 2 compares the aspartylation kinetics of wild-type tRNAAsp transcript in the absence and presence of the aminoglycoside tobramycin. The aspartylation of tRNAAsp decreases with increased concentrations of tobramycin (1–3 mM). The lines in the absence and presence of the antibiotic intercept the y-axis at one point, indicating that tobramycin acts as a competitive inhibitor with respect to tRNAAsp (Figure 2). The Michaelis–Menten parameters kcat and Km in the presence and the absence of tobramycin and/or total tRNA from yeast are summarized in Table I together with the relative kinetic specificity constants (kcat/Km)rel = (kcat/Km)tobramycin/(kcat/Km)–tobramycin. A more intuitive number is also included, namely the loss in aminoacylation efficiency caused by the antibiotic. The presence of tobramycin affects mainly the Km by factors up to 30-fold (for 3 mM tobramycin at an ATP:Mg2+ ratio of 5:15 mM), while the kcat is only decreased 2-fold. As a consequence the relative specificity constants are decreased and the loss in aminoacylation efficiency increases up to 60-fold. Further, aminoacylation experiments using fully modified yeast tRNAAsp reveal that tobramycin inhibits charging to the same extent, showing equivalent kinetic parameters for aminoacylation compared with those of the corresponding in vitro transcripts (data not shown). The addition of tobramycin at various times or directly before the start of the reaction by AspRS does not show a change in the aminoacylation kinetics (data not shown). Tobramycin does not exhibit a time dependence of the inhibition of the aminoacylation reaction of tRNAAsp. Thus, the kinetics measurements indicate that binding of tobramycin leads to spatial conformational changes of the tRNA, resulting in a reduced affinity of tRNAAsp for AspRS or a loss in transition state stabilization of the tRNAAsp–AspRS complex. Figure 2.Inhibition of the aspartylation reaction of tRNAAsp transcripts by tobramycin. The double reciprocal plot (Lineweaver–Burk) shows the initial velocity of the aspartylation reaction as a function of tRNAAsp concentration in the absence of tobramycin (circles) and in the presence of tobramycin at 1 (squares), 2 (diamonds) and 3 mM (triangles). Download figure Download PowerPoint Table 1. Kinetic parameters for aspartylation of yeast tRNAAsp transcripts with yeast AspRS in the absence or presence of tobramycin ATP/MgCl2 (mM) Inhibitor tobramycin (mM) Competitor tRNAtotal [mM] Km (nM) kcat (s−1) kcat/Km (relative) Loss of specificity (x-fold) 5/15a 0 – 44 0.66 1 1 5/15a 1 – 466 0.33 0.047 21 5/15a 2 – 945 0.38 0.027 37 5/15a 3 – 1190 0.30 0.017 60 5/15a 3 10 151 0.27 0.12 8 5/15a 3 30 115 0.21 0.12 8 5/15a 3 90 95 0.18 0.13 8 2/10b 0 – 732 0.31 0.028 35 2/10b 3 – 1285 0.58 0.03 33 a ATP:Mg2+ ratio of 1:3. b ATP:Mg2+ ratio of 1:5. To determine whether the inhibition is specific for the tRNAAsp–AspRS interaction, the effect of tobramycin on tRNAAsp was determined in the presence of an increasing excess of competitor tRNA. For this purpose tRNAAsp is incubated together with up to 2-fold excess of tRNAPhe over tobramycin. No recovery of the aminoacylation activity is observed after the addition of native tRNAPhe (Figure 3A). In the high concentration range of competitor tRNA an inhibition effect of the aminoacylation reaction is noticeable, probably due to the addition of high concentrations of charges and salt. Moreover, assays in the presence of high levels of total tRNA (3- to 50-fold excess over tobramycin) also resulted in no recovery of aspartylation activity (Table I). Further analysis of the kinetic parameters shows only small effects on the aspartylation reaction. A loss of specificity by a factor of 8 is observed, but remains essentially unchanged with increasing concentrations of total tRNA. Figure 3.Kinetic measurements of the competition of tobramycin binding to tRNAAsp by other tRNAs. (A) Influence of an excess of competitor tRNA, e.g. tRNAPhe at 0.02 and 0.1 mM (2-fold excess compared with tobramycin), on the aspartylation reaction of tRNAAsp in the absence (unfilled bars) and in the presence of 0.05 mM tobramycin (filled bars). (B) Competition of tobramycin upon binding to tRNAAsp and tRNAPhe within a native mixture of all yeast tRNAs. The level of aminoacylation is expressed as the charging activity for the aspartylation by yeast AspRS in the absence (squares) and presence of 0.3 mM tobramycin (triangles) and as a control for the phenylalanylation by yeast PheRS in the absence (filled circles) and presence of 0.3 mM tobramycin (filled triangles). Download figure Download PowerPoint The next question addressed was whether tobramycin can specifically inhibit tRNAAsp charging within a mixture of various tRNA families. The level of aspartylation and phenylalanylation within a fraction of total tRNAs was monitored in the absence and presence of tobramycin. Figure 3B reveals that tobramycin exhibits the same inhibition potential towards the aspartate system as shown for purified tRNAAsp (see Figure 2). The aspartyl ation reaction is inhibited by ∼20%. However, the phenylalanylation of tRNAPhe within the same pool is not affected (Figure 3B). Electrostatic interactions play a dominant role in aminoglycoside–RNA binding (Tor et al., 1998). If the positively charged aminoglycoside is in competition with divalent metal ions, inhibition of the tRNA charging reaction could be overcome by increased concentrations. In one series of experiments, the ATP:MgCl2 ratio was kept constant (1:3), while varying the magnesium ion concentration from 7 to 60 mM. Figure 4 illustrates that the aminoacylation reaction reaches its maximum at 10 mM MgCl2. In the presence of tobramycin this maximum is reduced and shifted to 15 mM MgCl2. Reaction mixtures containing >12 mM MgCl2 reveal a decrease in aminoacylation activity as well as a reduction in the inhibition effect caused by tobramycin. A second set of experiments was carried out under constant ATP conditions (5 mM) with increased MgCl2 concentrations ranging from 1 to 50 mM giving identical results (data not shown). Figure 4.Kinetic measurements of the competition of tobramycin binding to tRNAAsp by magnesium ions. Influence of magnesium ions on the aspartylation reaction of tRNAAsp in a buffer solution containing increasing concentrations of MgCl2 up to 60 mM, while keeping the ATP and MgCl2 at a constant ratio of 1:3, in the absence of tobramycin (squares) and in the presence of 0.3 mM tobramycin (circles). Download figure Download PowerPoint Tobramycin could bind specifically to the catalytic site of yeast AspRS as a structural analogue of ATP. To exclude this possibility, the concentration of ATP in the aspartylation reaction was reduced from 5 to 2 mM ATP (leading to a change in the ATP:MgCl2 ratio from 1:3 to 1:5). The effect of tobramycin on the aspartylation kinetics of tRNAAsp is compared before and after the change of the ATP:MgCl2 ratio (Figure 5A and Table I). A slight change in the initial velocity is observed in the absence of the antibiotic. However, in the presence of tobramycin the inhibitory effect is identical, showing that ATP is not competing with tobramycin for binding to the catalytic site of AspRS. Figure 5.Aspartyl-adenylate formation by AspRS in the presence of tobramycin. (A) The Lineweaver–Burk plot shows the kinetics of the aspartylation reaction in the absence of tobramycin in a buffer solution containing 5 mM ATP and 15 mM MgCl2 (1:3) (squares), or 2 mM ATP and 10 mM MgCl2 (1:5) (diamonds) and in the presence of 3 mM tobramycin at an ATP:MgCl2 ratio of (1:3) (circles) or (1:5) (triangles). (B) Influence of tobramycin on the [32P]PPi–ATP exchange reaction catalysed by AspRS. Download figure Download PowerPoint Aspartyl-adenylate formation by ApsRS is not inhibited by tobramycin During the first step in the aminoacylation reaction, AspRS forms the activated aspartyl-adenylate in the absence of tRNAAsp. A series of pyrophosphate exchange [PPi] experiments were designed to estimate the degree of inhibition during adenylate formation. AspRS was incubated with radioactively labelled pyrophosphate and aspartic acid in the presence and the absence of the aminoglycoside. The initial rate of the pyrophosphate exchange reaction at all three concentrations of tobramycin (0.3–30 mM) tested is at the same level and no difference at the level of pyrophosphate incorporation either in the absence or presence of the aminoglycoside is visible (Figure 5B). Therefore, tobramycin does not act as a structural analogue of ATP or an amino acid in the aminoacylation reaction and does not interact with the catalytic domain of the AspRS itself during the amino acid activation step. Ultraviolet absorbance melting experiments reveal a destabilization of the tertiary structure of tRNAAsp upon interaction with tobramycin Ultraviolet (UV) absorbance melting curves were performed to assess the effect of the tobramycin–tRNAAsp interaction on the overall structural stability of the tRNAAsp. In the presence of 3 mM MgCl2 the shape of the melting curve of tRNAAsp shows a sharp and symmetrical profile (Figure 6) indicating a highly cooperative melting transition in a quasi all-or-none manner. The calculated melting temperature (Tm) of 65°C and the shape of the transition is in agreement with published data (Puglisi et al., 1993). After the addition of 1 mM tobramycin the melting profile of tRNAAsp changes dramatically with a drop of the Tm of 10°C to 55°C. The Tm and the shape of the melting transition in the presence of the antibiotic resemble that of tRNAAsp in the absence of magnesium ions (Puglisi et al., 1993). This broad, non-cooperative transition indicates several melting domains related to the independent melting behaviour of the helical regions of the tRNA. Therefore, it appears that tobramycin destabilizes the functional tertiary conformation of the tRNAAsp and/or the tRNAAsp–AspRS complex including magnesium ions and the adenylate. Indeed, disruption of tertiary interactions in the core of tRNAAsp results in a decrease of (kcat/Km)rel (Puglisi et al., 1993). Figure 6.UV absorbance melting experiments of yeast tRNAAsp transcripts in the absence and presence of 1 mM tobramycin. Points are experimental. The calculated Tm is indicated by the arrows. Download figure Download PowerPoint Fluorescence anisotropy experiments In order to determine quantitatively the specificity and stoichiometry of ligand binding, the fluorescence anisotropy of fluorescein-labelled tobramycin (Tob–Fl) was measured. Figure 8 shows the change in fluorescence anisotropy as a function of tRNA concentration. The anisotropy value r (see Equation 1 in Materials and methods) for Tob–Fl in the absence of tRNA is (25.50 ± 0.17) × 10−3. Addition of tRNAAsp results in a hyperbolic increase in the fluorescence anisotropy to (27.31 ± 0.04) × 10−3 for completely bound Tob–Fl, with 1.2 molecules of Tob–Fl found to bind with high affinity and specificity to tRNAAsp with an R value of 0.99 (see Equation 3 in Materials and methods). Fitting the plot for tRNAAsp with a 1:1 stoichiometry reveals a dissociation constant of 267 nM (see Equation 2 in Materials and methods). An active tRNAAsp anticodon loop variant (Figure 7A) gives similar results (data not shown). An aminoacylation-inactive 3D-folding variant of tRNAAsp (tRNAAsp mutant D, Figure 7B) containing the identity elements for aspartylation, but with a disrupted 3D structure, was also titrated. The initial value of the fluorescence anisotropy of Tob–Fl does not change significantly with the addition of tRNAAsp mutant D. The inset in Figure 8 shows the titration of Tob–Fl with fully modified tRNAPhe. The plot displays a large and slightly sigmoidal increase in the fluorescence anisotropy to ∼(105 ± 0.08) × 10−3 at very high concentrations of tRNAPhe. Upon binding to tRNAPhe the fluorescence anisotropy is reduced dramatically, indicating that the fluorophore is constrained in some manner upon binding to the RNA. The affinity of Tob–Fl for tRNAPhe is much lower, with a KD of 6.0 μM and 1.73 bound molecules of Tob–Fl per tRNAPhe with an R value of 0.99. The KD is increased by a factor of 20 compared with that of the tRNAAsp transcript. Hill plot analysis using the tobramycin–fluorescein binding data with yeast tRNAPhe resulted in a slope value of 1, indicating the absence of a cooperative binding effect (data not shown). The fluorescence anisotropy change measured for tobramycin upon binding to tRNAAsp is rather low (Δr = 2 × 10−3), while in the presence of tRNAPhe a difference in Δr of 80 × 10−3 anisotropy units is observed. A tobramycin binding aptamer interacting with high affinity with Tob–Fl shows a moderate anisotropy change (Δr = 30 × 10−3) (Wang et al., 1996). In this case, tobramycin binds to the RNA deep groove centred about a stem–loop junction site. A portion of the bound tobramycin, ring III and partly II (Figure 1B), is encapsulated between the floor of the deep groove and a looped-out cytosine residue that forms a flap over the binding site in the complex with the aptamer (Jiang et al., 1997). The primary amino group is the site of attachment to the column in the SELEX experiment of the tobramycin aptamer (Wang et al., 1996) and also the site of labelling with the fluorescein dye in our experiments. This amino group on the ring system I reaches slightly out into the solution (Jiang et al., 1997). The moderate change in the anisotropy value in the case of the aptamer can be explained by its degree of freedom. The relatively low anisotropy of Tob–Fl bound to tRNAAsp indicates that the fluorescein is still very mobile. The fluorescein group attached to the tobramycin probably reaches out into the solution, while other amino groups of tobramycin seem to be responsible for the specific binding at the site of the tertiary interaction, possibly between the D and T domains. However, the high anisotropy value found for Tob–Fl bound to tRNAPhe indicates that the reporter dye must be immobilized to a high degree. Figure 7.Sequences of tRNA variants used in the fluorescence anisotropy measurements. The identity elements for aspartylation are shadowed. Sequence variations from tRNAAsp are highlighted in lower-case letters. (A) The yeast tRNAAsp mutant A is an active anticodon loop variant of tRNAAsp. The anticodon sequence shows a shift of the GUC-identity elements for AspRS, but is active in the aspartylation reaction (J.Pütz and R.Giegé, unpublished data). (B) The 3D structure of the yeast tRNAAsp mutant D is prevented from folding into the native L-shaped structure by the insertion of a series of adenine nucleotides in the D and T domains, which impairs aminoacylation. Download figure Download PowerPoint Figure 8.Fluorescence anisotropy measurements of tobramycin–tRNA complex formation. Fluorescence anisotropy (r) of fluorescence-labelled tobramycin (Tob–Fl; 10 nM) as a function of tRNAAsp (triangles), tRNAAsp mutant D (circles) or transcribed E.coli tRNAAsp (squares) concentration. The solid line is calculated by curve fitting to Equation 2. The tRNAAsp mutant D (sequence, Figure 7B) and E.coli tRNAAsp showed only non-specific binding at high concentrations of transcript. The inset displays the fluorescence anisotropy (r) of Tob–Fl solution (10 nM) as a function of native tRNAPhe (inverted triangles) concentration. The solid line is calculated by curve fitting to Equation 2. Please note that the buffer conditions are reduced due a high quenching effect (see Materials and methods). Download figure Download PowerPoint The yeast AspRS enzyme is known to aminoacylate both the yeast tRNAAsp and the tRNAAsp from E.coli. In contrast, the bacterial enzyme of AspRS binds both tRNAs with similar affinity but cannot charge yeast tRNAAsp. A crystal structure of an inactive complex between yeast tRNAAsp and E.coli AspRS has recently been published confirming the crucial role of the native and properly adapted tRNA three-dimensional structure (Moulinier et al., 2001). The sequence of yeast tRNAAsp differs from E.coli tRNAAsp in 35 positions, four of which are located in the D domain and six in the T domain with two positions located in the T loop directly. The fluorescence anisotropy binding assay is used to test for binding of Tob–Fl to E.coli tRNAAsp. In contrast to yeast tRNAAsp, the addition of E.coli tRNAAsp does not induce a significant increase in the initial value of the fluorescence anisotropy of Tob–Fl (Figure 8). This corresponds to the behaviour of the aminoacylation-inactive tRNAAsp mutant D (Figure 8) containing the identity elements for aspartylation, but with a disrupted 3D structure. Since Tob–Fl does not bind to E.coli tRNAAsp, inhibition of aminoacylation of yeast AspRS by tobramycin is not expected. This experiment demonstrates that the conformational change necessary for tobramycin binding to a tRNA depends on those parts of the tRNA sequence underlying the tertiary structure. To investigate further the effect induced by tobramycin binding to the tRNAAsp on its interaction with AspRS, tRNAAsp was labelled at the free 5′ terminus with the fluorescent dye erythrosin (tRNAAsp-Ery). The fluorescence anisotropy value of tRNAAsp-Ery is very high (38 ± 0.2 × 10−3) in the absence of AspRS (Figure 9). The fluorescent dye might be restricted in its rotational freedom due to some interaction with the single-stranded 3′ CCA terminus. Upon addition of AspRS the fluorescence anisotropy value dropped down to a minimum of (28 ± 0.16) × 10−3 at 100 nM AspRS. Upon complex formation between tRNAAsp and AspRS a conformational rearrangement occurs that allows the fluorescent dye more rotational freedom. At the end of the titration with AspRS the inhibitor molecule was added in three steps to the tRNAAsp–AspRS complex. The fluorescence anisotropy value increased back to its original value with increasing concentrations of tobramycin. If tobramycin is bound to tRNAAsp, AspRS can no longer bind to the tRNA and the fluorescent dye can fold back at its original position. When tobramycin was allowed to form a complex with tRNAAsp before titration with AspRS, no significant change in the fluorescence anisotropy value was observed during the whole titrational range. Thus, the tobramycin–tRNAAsp complex functions as a competitive inhibitor in the aminoacylation reaction by preventing the synthetase from binding to its substrate. Figure 9.Fluorescence anisotropy measurements of tRNAAsp-Ery–AspRS complex formation. Fluorescence anisotropy r of fluorescence-labelled tRNAAsp (tRNAAsp-Ery; 1 nM) as a function of AspRS (circles) and after preincubation of tRNAAsp with 30 mM tobramycin (triangles). In the first case increasing amounts of tobramycin are added at the final AspRS concentration to monitor the effect of the inhibitor on the tRNAAsp-Ery–AspRS complex. Download figure Download PowerPoint The set of fluorescence anisotropy measurements shows that the inhibitor acts by binding to the tRNA substrate of the enzymic reaction thereby forming an inhibitory complex. Aminoacylation kinetics were performed under oversaturating inhibitor concentrations. Under these conditions and a measured KD in the lower nanomolar range one can assume that the concentration of the inhibitory complex is equal to the concentration of the substrate, e.g. tRNAAsp. A detailed analysis shows that the inhibition constant KI using the standard relationship (Equation 4): is in the lower nanomolar range at 36 nM. If one assumes that tobramycin acts as an inhibitor directly on the enzyme, the calculated KI would increase into the upper micromolar range (125 μM), a value much higher than the measured affinity binding constant. Discussion Our steady-state kinetics of the aminoacylation reaction demonstrate that tobramycin specifically inhibits by a competitive mechanism the charging of tRNAAsp in vitro (Figure 2) even in the presence of non-cognate tRNAs (either a mixture of all native tRNAs or in the presence of tRNAPhe) (Figure 3). Tobramycin is in competition with magnesium ions (Figure 4), but not with ATP (Figure 5A). Tobramycin does not act at the level of the formation of activated aspartyl-adenylate by AspRS (Figure 5B). The dissociation constant measured by fluorescence anisotropy is in the nanomolar range (Figure 8). UV absorption melting curves indicate that tobramycin disrupts the native conformation of tRNAAsp (Figure 6). Tobramycin does not bind either to a mutated tRNAAsp, which does not display a tertiary structure, or to E.coli tRNAAsp with sequence variations in both the D and T domains (Figure 8). The non-native tobramycin–tRNAAsp complex acts as a competitive inhibitor of the aspartylation reaction (Figure 9). Chemical and enzymic footprinting studies confirm that the binding of tobramycin alters specifically the native 3D structure of yeast tRNAAsp (to be published elsewhere). Binding of tobramycin to a tRNA does not lead automatically to a conformational change interfering with the recognition of the tRNA identity elements by the cognate aaRS. Indeed, fluorescence anisotropy measurements show micromolar binding of tobramycin to yeast tRNAPhe (Figure 8), but the phenylalanylation of yeast tRNAPhe by PheRS is not inhibited by tobramycin (Figure 3B). Among aminoglycosides, neomycin B has been demonstrated to inhibit the phenylalanylation of E.coli tRNAPhe with an I50 (e.g. concentration at 50% inhibition of the aminoacylation reaction) in the upper micromolar range (Mikkelsen et al., 2001). Using a crude extract of the tRNA fraction higher concentrations of the aminoglycoside are needed for the inhibition of the aminoacylation reaction. In the yeast tRNAPhe–aminoglycoside crystal complex only one neomycin molecule is observed. Neomycin B is bound to the upper part of the anticodon stem. The same authors tested the inhibition of lead (Pb2+) cleavage of yeast tRNAPhe by various aminoglycosides. They concluded that neomycin B binds probably with two molecules to tRNAPhe with an I50 of 300 μM. Previously, several aminoglyco
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