Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation

AUTOR(ES)

Walter, Frank

FONTE

Oxford University Press

RESUMO

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.

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