The tRNA Specificity of Thermus thermophilus EF-Tu

Abstract

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)⋅GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from −11.7 kcal/mol for Val-tRNA^Glu to −8.1 kcal/mol for Val-tRNA^Tyr, clearly establishing EF-Tu⋅GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of k _off varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.