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High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure
Kazuo Watanabe1, Alan M Lambowitz
1Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, School of Biological Sciences, University of Texas at Austin, 78712, USA.
RNA (New York, N.Y.)
|July 27, 2004
Summary
Mobile group II introns use proteins like LtrA to splice RNA and move. This study reveals that LtrA’s binding to the DIVa RNA structure must be balanced for efficient intron mobility.
Area of Science:
- Molecular Biology
- RNA Biology
- Genetics
Background:
- Mobile group II introns utilize encoded proteins with reverse transcriptase and maturase activities.
- The Lactococcus lactis Ll.LtrB intron's protein, LtrA, binds to subdomain DIVa for splicing and mobility.
- LtrA binding to DIVa is crucial for translational regulation, RNA splicing, and intron mobility.
Purpose of the Study:
- To investigate the structural requirements of the LtrA binding site within intron subdomain DIVa.
- To determine the impact of specific base modifications within DIVa on LtrA binding and intron function.
- To elucidate the delicate balance of LtrA-DIVa interaction for RNA splicing and intron mobility.
Main Methods:
- In vitro selection assays to identify high-affinity LtrA binding sites.
- RNA structure probing and mutational analysis of subdomain DIVa.
- Assays to measure RNA splicing efficiency and intron mobility.
Main Results:
- An extended stem-loop structure with a bulged adenosine (A556) was identified as critical for tight LtrA binding.
- Altering A556 significantly inhibited intron mobility but had minor effects on splicing.
- Modifications at A553 resulted in tighter LtrA binding but also unexpectedly inhibited intron mobility.
Conclusions:
- LtrA binding to DIVa is finely tuned; both too weak and too tight binding impair intron mobility.
- The maturase/DIVa interaction shares similarities with bacteriophage coat protein/RNA hairpin interactions.
- Understanding this delicate balance is key to deciphering group II intron dynamics and regulation.