The involvement of the anticodon adjacent modified nucleoside N-[9-( -D-ribofuranosyl) purine-6-ylcarbamoyl]-threonine in the biological function of E.coli tRNAile

Abstract

TRNA ^ile was isolated from E. coli Cp 79 (leu ⁻ , arg ⁻ , thr ⁻ , his ⁻ , thiamin−, RC ^rel ) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(β-D ₌ -ribofuranosyl)-purin-6-ylcarbamoyl]threonine, t ⁶ Ado, than tRNA ^ile from cells grown on an optimum concentration of thr and containing a normal complement of t ⁶ Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3′-terminal dinucleotide, and to form an ile-tRNA ^ile -Tu-GTP complex. In contrast, the t ⁶ Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA. and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t ⁶ Ado-deficient ile-tRNA demonstrated no binding at Mg ^2$ concentrations ≤10 mM, while the normal ile-tRNA bound at low Mg ^2$ concentrations. Tetracycline had the same effect on the normal as on the t ⁶ Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t ⁶ Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t ⁶ Ado is required for proper codon-anticodon interaction.