Nucleotides that contribute to the identity of Escherichia coli tRNAPhe

Abstract A series of sequence variants of amber suppressor genes of tRNA Phe were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA Phe . The second type of variant involved total gene synthesis based on wild-type tRNA Phe sequences found in Bacillus subtilis and in Halobacterium volcanii . In the third type of variant, the identity of E. coli tRNA Lys was changed to that of tRNA Phe . The nucleotides which are important for tRNA Phe identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA Phe identity. The fourth nucleotide from the 3′ end of tRNA Phe has some importance for identity.