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The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity
1CNRS, Laboratoire d'Enzymologie et de Biochimie Structurales, Gif-sur-Yvette, France.
RNA (New York, N.Y.)
|July 22, 1998
Summary
Yeast pseudouridine synthase 1 (Pus1p) modifies multiple tRNA sites, including distinct noncontiguous positions within a single tRNA molecule. However, another enzyme modifies tRNA(Tyr) at position 35, indicating overlapping specificities in yeast tRNA pseudouridylation.
Area of Science:
- Molecular Biology
- RNA Modification
- Enzymology
Background:
- The yeast gene PUS1 encodes tRNA pseudouridine synthase 1 (Pus1p).
- Previous work showed Pus1p catalyzes pseudouridine formation at positions 34 and 36 in tRNA(Ile) in an intron-dependent manner.
Purpose of the Study:
- To investigate the substrate specificity and catalytic capabilities of yeast pseudouridine synthase 1 (Pus1p).
- To identify other pseudouridylation sites modified by Pus1p in vitro and in vivo.
- To determine the enzyme responsible for pseudouridine formation at position 35 in tRNA(Tyr).
Main Methods:
- In vitro assays using T7 transcripts of various yeast tRNA genes with recombinant Pus1p.
- Analysis of cell-free extracts from a PUS1-disrupted yeast strain.
- Mapping of pseudouridine residues in tRNAs from the PUS1-disrupted strain.
Main Results:
- Yeast Pus1p catalyzes pseudouridine formation at positions 27 and/or 28 in multiple cytoplasmic tRNAs.
- Pus1p modifies position 35 in intron-containing tRNA(Tyr) in vitro.
- A PUS1-disrupted strain lacks pseudouridylation at positions 27, 28, 34, and 36, but retains position 35 modification in tRNA(Tyr), indicating another enzyme's involvement.
Conclusions:
- Yeast Pus1p exhibits broad substrate specificity, modifying distinct noncontiguous sites within tRNA molecules.
- A separate tRNA pseudouridine synthase with overlapping specificity exists for position 35 in tRNA(Tyr).
- Pus1p is also implicated in vivo in modifications at U26, U65, and U67.