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Immobilized Tryptophanyl-tRNA Synthetase from Bacillus subtilis
Feng Xu1, Li Chen, Quan-Sheng Liu
1State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. yxjin@sunm.shcnc.ac.cn
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao Acta Biochimica Et Biophysica Sinica
|July 12, 2002
Summary
Immobilized tryptophanyl-tRNA synthetase (TrpRS) shows enhanced stability and is effective for SELEX, successfully isolating RNA structures similar to tRNA(Trp). This advances understanding of tRNA-protein interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Investigating the recognition mechanism between tRNA(Trp) and tryptophanyl-tRNA synthetase (TrpRS) is crucial for understanding gene expression.
- Studying the structure-function relationship of tRNA(Trp) and TrpRS provides insights into aminoacyl-tRNA synthetase activity.
Purpose of the Study:
- To purify and immobilize TrpRS from Bacillus subtilis for studying its properties.
- To evaluate the utility of immobilized TrpRS as an affinity matrix for SELEX (Systematic Evolution of Ligands by Exponential Enrichment).
- To explore the potential of SELEX in identifying RNA molecules that mimic tRNA(Trp) structure.
Main Methods:
- Purification and immobilization of TrpRS onto CNBr-activated Sepharose 4B.
- Characterization of the immobilized TrpRS, including thermal and shelf stability, optimum temperature, and pH.
- Application of the SELEX method using a randomized RNA library and immobilized TrpRS column selection over three rounds.
Main Results:
- Immobilized TrpRS exhibited significantly higher thermal and shelf stability compared to native TrpRS.
- The immobilized enzyme demonstrated improved operational stability with increased optimum temperature and pH.
- SELEX resulted in a substantial enrichment of RNA binders, increasing from 4.3% to 14.7% after three rounds.
- Sequencing revealed RNA structures with similarity to the acceptor stem of tRNA(Trp).
Conclusions:
- Immobilized TrpRS serves as a stable and effective affinity chromatography matrix.
- The SELEX method using immobilized TrpRS is suitable for isolating RNA molecules with tRNA(Trp)-like structures.
- This study enhances the understanding of tRNA-protein interactions and provides a tool for RNA aptamer selection.