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Tertiary core rearrangements in a tight binding transfer RNA aptamer
T L Bullock1, L D Sherlin, J J Perona
1Department of Chemistry and Biochemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California at Santa Barbara, 93106-9510, USA.
Nature Structural Biology
|July 6, 2000
Summary
Researchers engineered a mutant tRNA (transfer RNA) that binds to glutaminyl-tRNA synthetase (GlnRS) with 30-fold higher affinity. This enhanced binding resulted from stabilizing the tRNA
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Glutaminyl-tRNA synthetase (GlnRS) is essential for protein synthesis, utilizing specific tRNA (transfer RNA) molecules.
- In vitro selection experiments aim to develop high-affinity aptamers for GlnRS, improving our understanding of RNA-protein interactions.
Purpose of the Study:
- To engineer a mutant tRNA (tRNAGln) with enhanced binding affinity to its cognate enzyme, glutaminyl-tRNA synthetase (GlnRS).
- To elucidate the structural basis for improved RNA-protein binding affinity through cocrystallography.
Main Methods:
- In vitro selection to identify aptamers with high affinity for GlnRS.
- Site-directed mutagenesis to create a specific tRNA loop mutant (5'-44AGGU48-3').
- X-ray crystallography to determine the 2.7 Å cocrystal structure of the mutant tRNA-GlnRS complex.
Main Results:
- The engineered tRNA mutant exhibited a 30-fold increase in binding affinity for GlnRS compared to the wild-type tRNA.
- Cocrystal structure revealed significant rearrangements in the tRNA's central tertiary core, not the RNA-protein interface.
- The mutant tRNA core displayed a novel G15-U48 hydrogen bond, a sulfate binding pocket, and enhanced base stacking.
Conclusions:
- Enhanced protein binding to globular RNA can be achieved by stabilizing internal RNA tertiary interactions.
- Structural modifications in the tRNA core, rather than direct optimization of the RNA-protein interface, drive improved binding affinity.
- This study provides insights into RNA structural dynamics and their impact on enzyme recognition and binding specificity.