The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity

Abstract

We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of Ψ₃₄ and Ψ₃₆ in the anticodon loop of the yeast minor tRNA^Ile in vitro (Simos G et al., 1996, EMBO J 15:2270–2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA^Tyr (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (Ψ) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of Ψ₂₇, Ψ₂₈, Ψ₃₄, and Ψ₃₆ in vitro, however, Ψ₃₅ formation in the intron-containing tRNA^Tyr(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA^Tyr, another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U₂₆, U₆₅, and U₆₇ in vivo.