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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
A small ribozyme with dual-site kinase activity.
Elisa Biondi1, Adam W R Maxwell, Donald H Burke
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri School of Medicine, Columbia, MO 65211, USA. biondie@missouri.edu
Nucleic Acids Research
|May 24, 2012
Summary
Kinase ribozyme K28 catalyzes dual-site phosphorylation using a complex active site. This ribozyme
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Catalysis
Background:
- Nucleic acids can catalyze phosphoryl transfer reactions.
- Kinase ribozymes exhibit catalytic activity, including phosphorylation.
- The K28 ribozyme displays a complex active site structure.
Purpose of the Study:
- To investigate the dual-site phosphorylation activity of kinase ribozyme K28.
- To elucidate the structural basis for K28's complex catalytic mechanism.
- To map the phosphorylation sites and understand the active conformation.
Main Methods:
- Radiolabeling with [γ-(32)P]GTP and DNAzyme-mediated cleavage.
- Alkaline digestion and primer extension pausing for site mapping.
- Enzymatic digestion, mutational analysis, and nuclease sensitivity assays.
Main Results:
- K28 phosphorylates two distinct residues separated in sequence.
- A constrained pseudoknot structure with unusual connectivity was identified.
- Donor-induced folding was observed via altered nuclease sensitivities.
- Evidence suggests simultaneous dual-site phosphorylation within individual RNA strands.
Conclusions:
- Kinase ribozyme K28 possesses a unique active site for dual-site phosphorylation.
- A compact pseudoknot structure, not global rearrangement, underlies this activity.
- Local dynamics within the structure facilitate the complex catalytic mechanism.
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