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. 2002 Feb 15;21(4):760-8.
doi: 10.1093/emboj/21.4.760.

Binding of tobramycin leads to conformational changes in yeast tRNA(Asp) and inhibition of aminoacylation

Frank Walter  1 , Joern Pütz, Richard Giegé, Eric Westhof
Affiliations

Binding of tobramycin leads to conformational changes in yeast tRNA(Asp) and inhibition of aminoacylation

Frank Walter et al. EMBO J. .

Abstract

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 tRNA(Asp) transcript indicate that the complex between tRNA(Asp) and tobramycin is a competitive inhibitor of the aspartylation reaction with an inhibition constant (K(I)) of 36 nM. Addition of an excess of heterologous tRNAs did not reverse the charging of tRNA(Asp), 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-PP(i) 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 tRNA(Asp). Fluorescence anisotropy using fluorescein-labelled tobramycin reveals a stoichiometry of one molecule bound to tRNA(Asp) with a K(D) of 267 nM. The results indicate that aminoglycosides are biologically effective when their binding induces a shift in a conformational equilibrium of the RNA.

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Figures

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Fig. 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.
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Fig. 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).
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Fig. 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).
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Fig. 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).
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Fig. 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.
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Fig. 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.
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Fig. 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.
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Fig. 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).
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Fig. 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.
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Fig. 10. Scheme of inhibition of function by antibiotics. (A) Scheme of inhibition of aspartylation of yeast tRNAAsp by tobramycin. The L-shaped structure of yeast tRNAAsp is stabilized by magnesium ions and cationic polyamines. AspRS is able to recognize the spatial arrangement of the identity elements on the tRNA structure and binds to the native conformation of tRNAAsp. If the functional complex is formed, the tRNAAsp becomes aspartylated by AspRS. Tobramycin also binds with high affinity to tRNAAsp. Binding of tobramycin disrupts and destabilizes the native structure of tRNAAsp, and AspRS is unable to bind to the unfolded tRNAAsp conformation. Thus, the antibiotic, by interacting with the native tertiary structure of tRNAAsp, can interfere with the subsequent productive interaction of tRNAAsp with AspRS. (B) Conformational states of RNA molecules and interaction with ligands. In the native conformation (A) an RNA molecule can perform its natural function. Upon a conformational change, the RNA adopts either a non-native conformation or another conformation related to alternative function (B). Polyamines and specific divalent metal ions (e.g. magnesium ions) usually stabilize the native state of an RNA molecule. In contrast, inhibitors, antibiotics or appropriate cofactors may shift the equilibrium to either a non-native state of the RNA or an alternative conformation, thereby interfering with its function.
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