RNA Sequence and Two-dimensional Structure Features Required for Efficient Substrate Modification by the Saccharomyces cerevisiae RNA:Ψ-Synthase Pus7p
Résumé
The RNA:pseudouridine (Ψ) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U13 in several yeast tRNAs, U35 in the pre-tRNA Tyr (GΨA), U35 in U2 snRNA and U50 in 5S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Ψ-synthases found in Bacteria, Archaea and Eukarya. While several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of an 7 nt–long sequence including the modified U residue in all natural Pus7p substrates suggested importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis we designed a set of RNA variants derived from the yeast tRNA Asp (GUC), pre-tRNA Tyr (GΨA) and U2 snRNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U-2 and A +1 residues (as referred to modified U 0) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4,-3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5' to the target U 0 residue (position-1) is not important for efficient modification. Alteration of tRNA 3D structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence at least of one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification.
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