Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the TΨ-loop of yeast tRNAs

Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T-54) and pseudouridine (psi(55)). To identify the tRNA elements recognized by tRNA:m(5)uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA(Asp) mutants were used. Some variants contained point mutations, 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 in 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 that 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 longer stem is required for efficient T-54 formation. None of the conserved 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 canonical nucleotides. Yeast tRNA:m(5)uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. interestingly, in a tRNA(Asp) variant in which the T psi-loop was permuted Mi with the anticodon-loop. The new U-32 and U-33 residues derived from the T psi-loop were quantitatively converted to T-32 and psi(33), respectively. Structural mapping of this variant with ethylnitrosourea confirmed 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 local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m(5)uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase. (C) 1997 Academic Press Limited.