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Tertiary Base Triple Formation in the SRV-1 Frameshifting Pseudoknot Stabilizes Secondary Structure Components
Lixia Yang1,2,3, Desiree-Faye Kaixin Toh3, Manchugondanahalli S Krishna3
1School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, Sichuan 610054, P. R. China.
Biochemistry
|November 9, 2020
Summary
Minor-groove base triples in the simian retrovirus type 1 (SRV-1) frameshifting pseudoknot stabilize RNA structures. Disrupting these tertiary interactions enhances PNA invasion, revealing their crucial role in frameshifting efficiency.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot utilizes minor-groove base triples between stem 1 and loop 2 to stimulate -1 ribosomal frameshifting.
- The precise impact of these tertiary base triple formations on the local stability of secondary structures (stem 1 and stem 2) and overall frameshifting efficiency remains unclear.
Purpose of the Study:
- To investigate how tertiary base triple formation in the SRV-1 pseudoknot influences the stability of its secondary structural elements.
- To explore the relationship between pseudoknot mechanical stability, tertiary interactions, and ribosomal frameshifting efficiency using peptide nucleic acid (PNA) invasion assays.
Main Methods:
- Synthesis of short peptide nucleic acids (PNAs) designed to invade stem 1 and stem 2 of the SRV-1 pseudoknot.
- Nondenaturing polyacrylamide gel electrophoresis (PAGE) to assess PNA binding to the SRV-1 pseudoknot and its mutants.
- Analysis of PNA invasion to infer changes in pseudoknot stability and the role of tertiary interactions.
Main Results:
- Mutations disrupting the stem 1-loop 2 base triples in the SRV-1 pseudoknot led to enhanced invasion by both stem 1- and stem 2-targeting PNAs.
- These findings suggest that the tertiary stem 1-loop 2 base triple interactions stabilize both stem 1 and stem 2, indicating a long-range coupling effect.
- The dissociation constants of the PNAs correlated positively with pseudoknot mechanical stabilities and frameshifting efficiencies.
Conclusions:
- Tertiary base triple interactions within the SRV-1 pseudoknot play a critical role in stabilizing its secondary structural components through long-range effects.
- PNA invasion assays provide a valuable method for characterizing the energetic contributions of tertiary interactions and ligand binding in nucleic acid structures.
- Understanding these interactions is key to deciphering the mechanisms of ribosomal frameshifting and viral replication.
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