MAJOR IDENTITY DETERMINANTS FOR ENZYMATIC FORMATION OF RIBOTHYMIDINE AND PSEUDOURIDINE IN THE T-PSI-LOOP OF YEAST TRANSFER-RNAS

Almost all transfer RNA molecules sequenced so far contain two univers al modified nucleosides at positions 54 and 55, respectively: ribothym idine (T-54) and pseudouridine (psi(55)). To identify the tRNA element s recognized by tRNA:m(5)uridine-54 methyltransferase and tRNA:pseudou ridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 4 3 yeast tRNA(Asp) mutants were used. Some variants contained point mut ations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the T psi-loop with only one G . C base-pair as stem (9-mer). All substrates !full-sized tRNA(Asp) and various minihelices) were produced ill vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl-L-methionine as a methyl donor. The results indicate th at the minimal substrate for enzymatic formation of psi(55) is a stem/ loop structure with only four G . C base-pairs in the stem, while a lo nger stem is required for efficient T-54 formation. None of the conser ved nucleotides (G(53), C-56, A(58) and C-61) and U-54 for psi(55) or U-55 for T-54 formation can be replaced by any of the other three cano nical nucleotides. Yeast tRNA:m(5)uridine-54 methyltransferase additio nally requires the presence of a pyrimidine-60 in the loop. interestin gly, in a tRNA(Asp) variant in which the T psi-loop was permuted Mi wi th the anticodon-loop, I;he new U-32 and U-33 residues derived from th e T psi-loop were quantitatively converted to T-32 and psi(33), respec tively. Structural mapping of this variant with ethylnitrosourea confi rmed that the intrinsic characteristic structure of the T psi-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a T psi-loop structure. These results demonstrate that a l ocal conformation rather than the exact location of the U-U sequence w ithin the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m(5)uridine-54 methyltransferase and tRNA:ps eudouridine-55 synthase. (C) 1997 Academic Press Limited.