tRNA leucine identity and recognition sets.

Abstract Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli , tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA Leu function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA Leu variants with their type II counterparts. A minimum of six conserved tRNA Leu nucleotides were required to change the amino acid identity and recognition of a type II tRNA Ser amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary “Levitt base-pair” in tRNA Ser was changed to A15-U48; the number of nucleotides in the α and β regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA Leu ; a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 “discriminator” base of tRNA Ser was changed to A73. This minimally altered tRNA Ser exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA SerΔ ; an amber suppressor in which the tRNA Ser type II extra-stem-loop is replaced by a consensus type I loop). This “type I” swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap. The type I and II swaps revealed differences in the contributions of the tRNA Leu acceptor stem base-pairs to tRNA Leu function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA Leu acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.