Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating −1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA–RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1–loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1–loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.

中文翻译：

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SRV-1移假结中的第三纪三重形成稳定了二级结构成分。

在1型猿猴逆转录病毒（SRV-1）mRNA移码假结的茎1和环2之间形成的小沟碱基三元组对于刺激-1个核糖体移码至关重要。第三碱基三重形成如何影响二级结构（茎1和茎2）的局部稳定性，从而对核糖体移码效率尚不十分了解。我们制备了一种短肽核酸（PNA），有望通过PNA-RNA双链体形成侵入SRV-1假结的茎1，从而通过翻译核糖体模拟茎1的展开过程。此外，我们使用PNA入侵SRV-1假结中的茎2。我们的PNA与SRV-1假结和突变体结合的非变性聚丙烯酰胺凝胶电泳数据显示，环2中的突变破坏了SRV-1假结中环2和茎1之间的碱基三重形成，导致两个PNA的侵袭增强。我们的数据表明，SRV-1假结中的三级茎1环2基三重相互作用可以稳定茎1和茎2的二级结构成分。茎2的稳定性因此与茎1环2的结构稳定性相关。通过远程作用介导的碱基三倍。两个PNA的表观解离常数与假结机械稳定性和移码效率正相关。