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Published on: February 16, 2015
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In vitro characterization of Dhr1 from Saccharomyces cerevisiae
Ran Lin1, Carl C Correll2, Arlen W Johnson1
1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, United States.
Methods in Enzymology
|August 14, 2022
Summary
The essential enzyme Dhr1 unwinds U3 RNA from pre-ribosomes, enabling the formation of the central pseudoknot crucial for small ribosomal subunit assembly. This study details Dhr1 purification and activity assays.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The cytosolic small ribosomal subunit (SSU) assembly is vital for protein synthesis.
- Dhr1 (S. cerevisiae) is an essential RNA helicase involved in SSU biogenesis.
- U3 small nucleolar RNA (snoRNA) transiently inhibits the formation of the central pseudoknot in pre-ribosomal RNA (rRNA).
Purpose of the Study:
- To elucidate the role of Dhr1 in facilitating the central pseudoknot formation during SSU assembly.
- To provide detailed protocols for the purification and characterization of Dhr1's enzymatic activities.
Main Methods:
- Purification of recombinant Dhr1 protein.
- In vitro assays to measure RNA-dependent ATPase activity.
- RNA unwinding assays to assess Dhr1's helicase function.
Main Results:
- Purified Dhr1 exhibits RNA-dependent ATPase activity.
- Dhr1 demonstrates specific RNA unwinding activity, consistent with its role in U3 snoRNA displacement.
- Protocols for Dhr1 purification and activity assays are established.
Conclusions:
- Dhr1 is a key enzyme that resolves RNA-DNA structures by unwinding U3 snoRNA from pre-rRNA.
- The established protocols enable further investigation into Dhr1's mechanism and regulation.
- Understanding Dhr1 function is critical for comprehending ribosome biogenesis and its associated pathologies.
