Single-Molecule Study of a Frameshift-Stimulating RNA Pseudoknot Structure
Yu-Ting Chen1, Kai-Chun Chang1, You-Hsin Lin1, Yi-Lan Chen1, Chiung-Fang Hsu1, Cheng-Fu Chang1, Jin-Der Wen1*
1Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
* presenting author:Jin-Der Wen,
Proteins are synthesized by ribosomes in the cell in a process called translation. The ribosome reads consecutive tri-nucleotide codons on the messenger RNA frame. Minus-one frameshifting occurs when the third nucleotide of a codon is reread for the following codon. This is commonly used to regulate the relative expression levels of two proteins in many bacteria and viruses. An RNA structure, mostly a pseudoknot or a hairpin, is required to efficiently stimulate -1 ribosomal frameshifting. Here we used a model frameshift-stimulating RNA pseudoknot, named DU177, to study how the structural features, including the conserved adenosine stretch in Loop 2, affect frameshifting. By using optical tweezers, a single-molecule technique, we found that the mechanical stability of DU177 was decreased when the adenosine stretch was changed to pyrimidines, and the frameshifting efficiency was concomitantly decreased. We then used an RNA hairpin annealed to an RNA oligomer to mimic the DU177 pseudoknot conformation. The hairpin stability was greatly increased in the presence of adenosine stretch and the reformation of the base triples that were originally involved in stabilization of the pseudoknot. Consistent results were obtained when we applied single-molecule FRET to measure the conformation of the RNA oligomer. Interestingly, mutations that disrupted the base triples distal from the adenosine stretch also broke the nearby base triples, resulting in disruption of the interaction between the adenosine stretch and the hairpin. Our results provide insights into the formation dependence of tertiary interactions in a frameshift-stimulating RNA pseudoknot.

Keywords: single-molecule, optical tweezers, FRET, frameshift