Binding of tobramycin leads to conformational changes in yeast tRNA(Asp) and inhibition of aminoacylation.
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.
Walter
Frank
F
UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, F-67084 Strasbourg Cedex, France.
Pütz
Joern
J
Giegé
Richard
R
Westhof
Eric
E
D000215:Acylation; D001483:Base Sequence; D002240:Carbohydrate Sequence; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014031:Tobramycin / Q000378:metabolism*
D012331:RNA, Fungal; D012349:RNA, Transfer, Asp; D014031:Tobramycin