RNA sequence and two-dimensional structure features required for efficient substrate modification by the Saccharomyces cerevisiae RNA:{Psi}-synthase Pus7p.
The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Psi-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the 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)(GPsiA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U(-2) and A(+1) residues (nucleotide numbers refer to target 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 three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification.
Urban
Alan
A
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, CNRS-UHP Nancy I, Nancy Université, 54506 Vandoeuvre-les-Nancy Cedex, France.
Behm-Ansmant
Isabelle
I
Branlant
Christiane
C
Motorin
Yuri
Y
D001483:Base Sequence / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012322:RNA Precursors / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; C474390:Pus7 protein, S cerevisiae; D012322:RNA Precursors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; D012343:RNA, Transfer