tRNA leucine identity and recognition sets

Transfer RNAs(tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting gr oups are classified as type II. Leucine tRNAs comprise one such group. We u sed both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. Tn addition, to investigate the role o f the tRNA fold, we compared the in vivo and in vitro characteristics of ty pe I tRNA(Leu) variants with their type II counterparts. A minimum of six conserved tRNA(Leu) nucleotides were required to change th e amino acid identity and recognition of a type II tRNA(Ser) amber suppress or from a serine to a leucine residue. Five of these nucleotides affect tRN A tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta reg ions of the D-loop was changed to achieve the positioning of G18 and G19 th at is found in all tRNA(Leu) a base was inserted at position 47n between th e 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) ex clusively 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(Ser Delta); 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 tR NA(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 acceptor stem sequence. The type I and II tRNAs used in this stud y differed only in the sequence and structure of the variable loop. By alte ring this loop, and thereby possibly introducing subtle changes into the ov erall tRNA fold, it became possible to detect otherwise cryptic contributio ns of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed. (C) 2000 Academic Press.