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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Published on: June 16, 2011
Selection of viral RNA-derived tRNA-like structures with improved valylation activities
1Abteilung Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Göttingen, Germany.
Biochemistry
|May 23, 2000
Summary
Turnip yellow mosaic virus (TYMV) RNA mimics tRNA structure for valylation. In vitro selection revealed variants with improved valylation efficiency, suggesting natural viral sequences prioritize other evolutionary pressures.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The turnip yellow mosaic virus (TYMV) tRNA-like structure (TLS) mimics tRNA L-shape and is recognized by yeast valyl-tRNA synthetase (ValRS).
- The TYMV TLS possesses a noncanonical 3'-terminus with an anticodon loop and a pseudoknotted amino acid accepting domain.
Purpose of the Study:
- To verify the complete valine identity set for TYMV TLS.
- To determine the impact of the pseudoknot on valylation efficiency.
- To investigate functional communication between the anticodon and amino acid accepting domains.
Main Methods:
- In vitro selection of valylatable RNA variants from a randomized TYMV TLS pool.
- Randomization targeted anticodon loop nucleotides and pseudoknot loop L1 length and sequence.
- Nine rounds of selection followed by isolation and sequencing of 42 variants.
Main Results:
- 17 RNA variants were efficiently charged by yeast ValRS.
- Conserved nucleotides were identified in the anticodon loop (A56, C55) and 3'-end (C53).
- Selected variants showed improved valylation efficiency (5-50x) compared to wild-type TYMV TLS.
Conclusions:
- The natural TYMV TLS sequence may not be solely optimized for aminoacylation efficiency.
- Evolutionary pressures beyond aminoacylation likely shaped the natural viral sequence.
- The study provides insights into RNA structure-function relationships and viral evolution.
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