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Updated: Jun 12, 2026
11:13
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Convergent donor and acceptor substrate utilization among kinase ribozymes
Elisa Biondi1, David G Nickens, Samantha Warren
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA. biondie@missouri.edu
Nucleic Acids Research
|June 1, 2010
Summary
Kinase ribozymes were selected to study donor nucleotide recognition for (thio)phosphorylation. These ribozymes show high specificity, primarily recognizing the nucleobase and phosphates, unlike typical aptamers.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Catalysis
Background:
- Kinase ribozymes catalyze phosphoryl group transfer, but donor nucleotide recognition and structural requirements for internal 2'-hydroxyl phosphorylation remain poorly understood.
- Systematic studies on kinase ribozyme donor recognition are lacking, hindering a deeper understanding of their catalytic mechanisms.
Purpose of the Study:
- To investigate donor nucleotide recognition by kinase ribozymes.
- To elucidate the structural requirements for 2'-hydroxyl (thio)phosphorylation by novel ribozymes.
- To identify consensus structural motifs in kinase ribozyme active sites.
Main Methods:
- Selection of novel self-phosphorylating ribozymes using ATP(gammaS) or GTP(gammaS).
- Kinetic analysis, donor nucleotide recognition assays, and secondary structure determination.
- Competition assays with nucleotide analogs and comparative structural analysis.
Main Results:
- Eight independent sequence families of kinase ribozymes were identified.
- Ribozymes exhibited high specificity for their cognate donors, with critical recognition elements including the Watson-Crick face of the nucleobase and donor phosphates.
- Phosphorylation sites were predominantly on unpaired guanonsines within internal bulges, and three consensus structural motifs were identified.
Conclusions:
- Kinase ribozymes utilize specific interactions, including phosphate binding, to orient the gamma phosphate for catalysis.
- Donor nucleotide recognition is primarily mediated by the nucleobase and phosphates, with minimal contribution from the Hoogsteen face.
- The identified structural motifs provide insights into the active site architecture of kinase ribozymes.
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