Upstream Structures Modulate the Folding of Frameshift-Stimulating RNA Pseudoknots

Shuo Wang¹, Yu-Tong Huang¹, Yu-Han Wu¹, Jui-Yun Tu¹, Jin-Der Wen^1*

¹Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

* Presenter:Jin-Der Wen, email:jdwen@ntu.edu.tw

SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic beginning in 2019. Its RNA genome contains a specific pseudoknot (PK) structure that stimulates programmed -1 ribosomal frameshifting (-1 PRF), a translational regulatory mechanism essential for the production of key viral proteins. The PK consists of three stems (S1, S2, and S3) connected by loops and has been shown to fold sequentially in the order of S1, S3, and S2. However, a recent study reported that the first half of S1 can alternatively form a stem (AS1) with a sequence upstream of the PK. In this study, we use optical tweezers to investigate how AS1 influences the folding of the PK. As expected, in the absence of the upstream sequence, the RNA predominantly folds into the PK structure in the S1-S3-S2 order, though the resulting PK conformations are highly heterogeneous. When a loop-truncated version of AS1 is introduced, PK formation remains efficient, but the conformational heterogeneity is modestly reduced. Furthermore, when AS1 is engineered to form a shorter but stable stem, the resulting PK conformations become more homogeneous. These findings suggest that overlapping secondary structures upstream of the pseudoknot can influence its folding pathway and conformational landscape. The biological significance of this modulation is currently under investigation.

Keywords: Single molecule, optical tweezers, RNA structures