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- Title
Binding of tobramycin leads to conformational changes in yeast tRNA<sup>Asp</sup> and inhibition of aminoacylation.
- Authors
Walter, Frank; Pütz, Joern; Giegé, Richard; Westhof, Eric
- 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 tRNAASP and tobramycin is a competitive inhibitor of the aspartylation reaction with an inhibition constant (K1) 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.
- Publication
EMBO Journal, 2002, Vol 21, Issue 4, p760
- ISSN
0261-4189
- Publication type
Academic Journal
- DOI
10.1093/emboj/21.4.760