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Yeast lariat debranching enzyme. Substrate and sequence specificity
K Nam1, R H Hudson, K B Chapman
1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
The Journal of Biological Chemistry
|August 12, 1994
Summary
Yeast RNA lariat debranching enzyme was purified and characterized. This enzyme efficiently degrades branched nucleic acids and shows a preference for purines, aiding future mechanistic studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- RNA lariat debranching enzyme is crucial for RNA processing.
- Understanding its function requires purified enzyme and characterized activity.
Purpose of the Study:
- Purify yeast RNA lariat debranching enzyme using bacterial expression.
- Characterize its substrate specificity and biochemical parameters.
- Develop assays for mechanistic and structural studies.
Main Methods:
- Bacterial overproduction and purification of the enzyme.
- Assessing activity on various branched nucleic acid substrates (group II introns, msDNAs, synthetic RNAs).
- Development of a trinucleotide release assay for kinetic analysis.
Main Results:
- The enzyme was purified to near homogeneity.
- It efficiently digests group II intron lariats, multicopy single-stranded DNAs (msDNAs), and synthetic branched RNAs.
- A preference for purines at the 2'-position of substrates was observed, consistent with native substrates.
Conclusions:
- The purified yeast RNA lariat debranching enzyme is catalytically active on diverse branched nucleic acids.
- Biochemical characterization reveals substrate preferences relevant to its biological role.
- Synthetic substrates facilitate future mechanistic and structural investigations of this enzyme.