A 2-THIOURIDINE DERIVATIVE IN TRANSFER RNA(GLU) IS A POSITIVE DETERMINANT FOR AMINOACYLATION BY ESCHERICHIA-COLI GLUTAMYL-TRANSFER RNA-SYNTHETASE

Early investigations into the interaction between Escherichia coli glu tamyl-tRNA synthetase (GluRS) and tRNA(Glu) have implicated the modifi ed nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first posit ion of the anticodon as an important contact for efficient aminoacylat ion. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of th e modification or simply involved other anticodon loop-nucleotides, no w occluded from interaction with the synthetase. Unmodified E. coli tR NA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (k(cat)/K(M)) compared to that of tRNA(Glu) prepa red from an overproducing strain. Through the use of recombinant RNA t echnology, we created several hybrid tRNAs which combined sequences fr om the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoac ylation of these hybrid tRNA molecules and of tRNAs with base substitu tions at positions of nucleotide modification, we show conclusively th at the modified uridine at position 34 in tRNA(Glu) is required for ef ficient aminoacylation by E. coli GluRS. This is only the second examp le of a tRNA modification acting as a positive determinant for interac tion with its cognate aminoacyl-tRNA synthetase.