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In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB endonuclease
V K Jayasena1, D Brown, T Shtatland
1Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347, USA.
Biochemistry
|February 20, 1996
Summary
T4 RegB endonuclease cleaves RNA at specific sites. Researchers used SELEX to find RNA molecules that enhance this cleavage, particularly with the help of Escherichia coli ribosomal protein S1.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- T4 RegB endonuclease cleaves specific -GGAG- sites in T4 bacteriophage RNA, affecting gene expression.
- RegB exhibits low activity in vitro, but this is significantly enhanced by Escherichia coli ribosomal protein S1.
Purpose of the Study:
- To identify RNA molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1.
- To understand the role of S1 in enhancing RegB activity.
Main Methods:
- Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was employed on a combinatorial RNA library.
- Selected RNA molecules were analyzed for their ability to be cleaved by RegB, with and without S1.
Main Results:
- SELEX yielded RNA molecules containing the -GGAG- recognition site, enriched in adenosine and cytosine nucleotides.
- Most selected RNAs required S1 for efficient RegB-catalyzed cleavage, demonstrating S1-dependence.
- A subset of RNAs showed S1-independent cleavage by RegB, albeit at lower rates.
Conclusions:
- Specific RNA ligands can be evolved to enhance T4 RegB endonuclease activity.
- Escherichia coli ribosomal protein S1 plays a crucial role in potentiating RegB-mediated RNA cleavage.
- The SELEX approach is effective in discovering functional RNA molecules interacting with specific endonucleases.